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					Vol. 7, 1 187-1 198, September                 1996                                                                                                                    Cell Growth & Differentiation              1187




Induction  of a Less Aggressive Breast Cancer                                                                                                             Phenotype                              by
Protein Kinase C-or and -3 Overexpression1


Andrea          Manni,2 Elizabeth Buckwalter,                                                            phosphorylation    of target proteins    (1 2). Considerable    ,evi-
Ransome           Etindi, Susan Kunselman,                                                               dence suggests    an important     role of PKC in breast cancer.
Anthony          Rossini,  David Mauger,  David Dabbs,                                          and      PKC expression     has been found to be increased          in breast
Laurence           Demers                                                                                tumors compared      with normal breasts (3). In addition,      PKC
Departments     of Medicine [A. M., E. B., A. E.] and Pathology                                          activity  has been shown to be associated        with negative ER
[S. K., A. A., D. M.j and Center for Biostatistics and Epidemiology
[D. D., L D.], The Pennsylvania                State     University       College    of Medicine,
                                                                                                         status (4) and the presence of EGF receptors         (5), thus sug-
Hershey,  Pennsylvania   17033                                                                           gesting      a correlation           with       an      aggressive         phenotype.            It is
                                                                                                         increasingly    recognized      that PKC isoenzymes      differ with re-
                                                                                                         gand to tissue expression         (normal versus malignant),      regula-
Abstract
                                                                                                         tion, substrate    specificity,    and biological function (1 2). Re-                     ,
To address the isoenzyme-specific       involvement of
                                                                                                         cent evidence       has pointed       to the possible     physiological
protein kinase C (PKC) in breast cancer       biology,
                                                                                                         importance         of differential           expression             and   subcellulan          local-
hormone-responsive       MCF-7 breast cancer      cells were
                                                                                                         ization of the various PKC isoenzymes    in breast cancer (6-
infected    with either PKC-a or -fi. cDNAs subcloned in
                                                                                                         8). The hormone-responsive    MCF-7   breast   cancer  cell line
the retroviral             expression           vector         pMV7.        Several        stable
                                                                                                         has been      shown        to express            both     calcium-dependent                   (a and
clones of PKC-overexpressing   cells were generated.
                                                                                                         3II) and     -independent              and i) isoforms
                                                                                                                                              (6,    #{128},
                                                                                                                                                          ,        (7, 8). Acqui-
Western analysis revealed cross-regulation  between
                                                                                                         sition of the multidnug-resistant        phenotype    by MCF-7 cells is
the a and 1 isoforms,  because induction of
                                                                                                         characterized     by a selective increase in PKC-a, whereas the
overexpression                 of one up-regulated   the other.
                                                                                                         expression     of PKC-8 and -#{128} is reduced and abolished,            re-
Overexpression                 of the a and 13 isoenzymes,    on the
                                                                                                         spectively    (7). These findings        indicate  that specific     alter-
other    hand, did not affect the                         already high endogenous
                                                                                                         ations in PKC isoenzyme          profiles have profound       influences
expression     of the novel 8, e, ,                         and isoforms.
                                                                                                         on   important        biological           properties         of      breast    cancer         cells,
Compared              with     control        clones,         PKC-a-          and     -fi-               such as drug resistance.    In support of this contention,    Ways
overexpressing                 MCF-7 cells exhibited                       more drastic
                                                                                                         et aL (8) recently showed that induction of overexpression       of
morphological                 changes         in response                to phorbol
                                                                                                         PKC-a in MCF-7 cells induced tumor progression           to a more
12-myristate               13-acetate          administration                characterized
                                                                                                         aggressive    phenotype.
by cellular flattening                   and vacuolization.                  More
                                                                                                             In these experiments,  we used a transfection    approach    to
importantly,       induction     of PKC-a and -p overexpression
                                                                                                         directly test the influence of overexpression      of PKC-a and
induced      a less aggressive        biological     behavior, which
was characterized            by reduced in vitro invasiveness and                                        _p1 on breast        cancer         phenotypic            features.       Our   results        dem-
                                                                                                         onstnate that there is cross-regulation      between the a and j3
markedly       diminished      tumor formation         and growth in
                                                                                                         isoforms in MCF-7 cells. In addition,     we also show that the
nude mice. These in vivo findings                can probably     best be
                                                                                                         coordinate  overexpression      of PKC-a and -p favorably     influ-
explained       by the dramatic       down-regulation        of estrogen
                                                                                                         ences the biological    behavior of breast cancer cells, with a
receptor      levels observed       in tumors      derived  from PKC-
                                                                                                         marked reduction     of in vivo tumongenesis      in nude mice.
a-infected       MCF-7 cells. Our data clearly show that it is
possible        to induce a less aggressive breast cancer
                                                                                                         Results
phenotype         by altering PKC isoenzyme expression.
                                                                                                         Biochemical        Properties       of PKC-overexpressing           MCF-7
                                                                                                         Breast      Cancer    Cells. Figs. 1 and 2 illustrate the kinase
Introduction
                                                                                                         activities of some of our clones of MCF-7 cells infected with
The PKC3 family of phospholipid-dependent                                          senine and thre-      either the 1 (Fig. 1) or a (Fig. 2) isoenzyme.          As can be seen,
onine       kinases          influence        diverse         cellular      functions         through
                                                                                                         the kinase activities measured in these clones were several-
                                                                                                         fold higher than those expressed            by the vector only-infected
                                                                                                         cells. These clones appear to be very stable, because they
Received   3/28/96;   revised 6/14/96; accepted 6/29/96.
The costs of publication of this article were defrayed in part by the                                    have been retaining         the same degree of ovenexpression             for
payment of page charges. This article must therefore be hereby marked                                    more than 2 years.
advertisement    in accordance   with 18 U.S.C. Section  1734 solely to mdi-
                                                                                                             Next, we analyzed          the PKC isoenzyme         profiles of our
cate this fact.
1   This work   was supported            by Grant       P01    CA4001       1-10 from     the National   clones by Western blot analysis. The results are shown in
Cancer Institute.                                                                                        Figs. 3 and 4 and document            the success of our infection. The
2 To whom      requests for reprints should be addressed, at Division of
Endocrinology,     Hershey Medical Center, 500 University  Drive, P.O. Box
                                                                                                         endogenous       expression       of the a and p isoforms        in control
850, Hershey,     Pennsylvania  17033. Phone: (717) 531-8395;    Fax: (717)                              and vector only-infected           cells was modest and absent, re-
531-5726.                                                                                                spectively.    Importantly,      our data show for the first time that
3 The abbreviations           used are: PKC, protein kinase C; ER, estrogen recep-
tor; EGF, epidermal           growth factor TGF, transforming growth factor; PMA,                        induction        of PKC-           ovenexpression              is associated            with      up-
phorbol     12-myristate        13-acetate;       dH2O, distilled         water;    E2, estradiol.       regulation       of the a isoform              (Fig.     3). The      reverse    cross-negu-
I 188   PKC Isoenzymes                    and Breast Cancer Phenotype




                                                                                                                                                       Positive             Wild

                                                                                                                                                       Control              Type                JV4                                         JD6


                                                                                                                                     a                                                                  S                                               4




                                                                                                                                                                                                                          -
                                                                                         H
                                                                                                                                                                                                                -




                                                                                                                                     o
                                            JV2            JA2       JA5         JB1      JC1      JC3      JD6


                                                                                Clone

        Fig. 1 . PKC activity in representative          clones of MCF-7 cells transfected
        with the 13 cDNA (JA2,          JA5, JB1, JC1, JC3, and JD6) or the vector only
        (JV2). Kinase activity was measured           as described  in “Materials and Meth-
        ods.” Similar results were obtained
        peated on numerous           occasions
                                                       when these measurements       were re-
                                                 over a time point in excess of 2 years to
                                                                                                                                      El                                !_#{149}!                                    -
        include     additional    clones besides     those shown.    PKC activity   in vector
        only-infected       clones was similar to that of wild-type     MCF-7 cells.



                             60’                                                                                                      c




                   E
                 5.-..

                   0
                   E
                   0
                             :                                                                                                       11                               rm-
                   >-.       30                                                                                                      Fig. 3. Representative        Western       immunoblots         of wild-type,      vector only-
                                                                                                                                     infected   (JV4), and j31 -infected      (JB1 and JD6) MCF-7 cells (see “Materials
                                                                                                                                     and Methods”       for details).     Western      blots were probed           with antibodies




                                                                                                                n
                   U         20
                   Cs                                                                                                                specific  for the different      isoforms     (indicated     to the left). Positive      control
                                                                                                                                     tissues included     rat brain homogenates            (a, /3, and ), mouse macrophage
                   Cs
                             10                                                                                                      lysate (6), HeLa cell lysate (#{128}), A-431 cells (ii). Western
                                                                                                                                                                             and                                       immunoblots
                   a
                                                                                                                                     were repeated     numerous       times to include additional          clones besides those

                               C-        n                    _             -             -           -
                                                                                                                                     depicted    with virtually identical
                                                                                                                                     able in the wild-type        MCF-7
                                                                                                                                                                               results. The a isoform was barely detect-
                                                                                                                                                                             cells and control          clones,    whereas      the /3
                                          1V2                 1A2           1A3           1A4         1A5          1A6               isoform was consistently         absent. The H isoform was not expressed                 by any
                                                                                                                                     of our clones (data not shown).
                                                                           Clone

        Fig. 2. PKC activity in representative        clones of MCF-7 cells transfected
        with the r cDNA (lA2-1A6) or the vector only (1V2). Kinase activity           was                                              Biological Properties    of PKC-overexpressing       MCF-7
        measured     as described     in “Materials    and Methods.”   Consistency     and                                           Breast Cancer Cells. Initially, we analyzed   the basal prolif-
        stability of PKC overexpression       over time was as described    in the legend
                                                                                                                                     erative         activities      in liquid       culture           of control             and     PKC-overex-
        to Fig. 1.
                                                                                                                                     pressing            MCF-7         cells     in the              presence             of different           serum
                                                                                                                                     concentrations                 (Fig. 5). As can be seen,                            no major        changes            in
                                                                                                                                     cell growth   were induced                       by infection   with either the l (Fig.
        lation           was         likewise     observed          (Fig.       4), as also          recently        shown           SA) or a (Fig. SB) isoform.                      Sensitivity  to the proliferative effect
        by Ways                et a/. (8). Among             the novel            PKCs          tested,     the 5,         #{128},
                                                                                                                                ,    of TGF-a              was      likewise        similar           in control              and     PKC-overex-
        and    isoforms                    were abundantly    expressed   by MCF-7 breast                                            pressing           breast       cancer         cells      (Fig.        6). Administration                 of PMA
        cancer    cells,                 and their expression     did not appear  to be al-                                          exerted          a marked         antiproliferative                effect,          which       was      similar       in
        tered             by PKC-a   and             -13 up-regulation                      (Figs. 3 and 4). We                      control         and     PKC-overexpressing                         MCF-7              clones       (Fig. 7).
        were             unable to detect               the expression                   of PKC-O in either our                           Striking         morphological               changes           were             induced         by PKC-cs
        control  or overexpressing                           clones             (data not shown).                                    and    -/3 overexpression   following                              the administration                     of PMA
           Because   treatment     with                      phonbol             esters has been                shown          to    (Figs.    8 and 9). As can be seen,                               PKC-overexpressing                        breast
        down-regulate                 ER expression     in MCF-7   cells (9), we meas-                                               cancer  cells              appeared    to be considerably       flatter and larger
        ured             cytosolic    ER levels in our clones.     As can be seen in                                                 than controls               (Figs. 8 and 9, compare       right   and /eft panels).
        Table             1 , ER expression    was retained   in PKC-cs-infeeted  cells,                                             We were             also      intrigued        by the           appearance                  of large       cellular
        whereas                    it was       somewhat          decreased                   in PKC-31           -infected          vacuoles,             which     were      most         prominent               following           PMA     admin-
        clones.              It is unlikely,         however,         that        this        reduction      has major               istration         in PKC-cs-infected                    cells     (Fig.        8) and          in both     control
        biological                   consequences,            because              the         reduced          ER levels            and PKC-l-infected         cells treated                                with this compound                      (Fig.
        should   still                be sufficiently         high         to allow            estrogen           action       to    9). Electron    microscopy      revealed                               that these vacuoles                     were
        take place.                                                                                                                  composed               of extracellular                matrix       and        were        lined      by the cell
                                                                                                                                                                                                                  Cell Growth          & Differentiation          1189




              Positive                     Wild                                                                                   Table        1     ER levels         in PKC-a-            and -/31-infected            MCF-7       breast      cancer
                                                                                                                                  cells
              Control                      Type                 1V2                 IA2                   IA6
                                                                                                                                     Logarithmically        growing     control   and PKC-overexpressing             MCF-7 cell
                                                                                                                                  clones     were plated        in the presence       of regular     growth    medium.      Near

a                          ::                                                                                                     confluency,      steroid-depleted
                                                                                                                                  ods”) were introduced
                                                                                                                                                                          culture conditions
                                                                                                                                                                    for an additional
                                                                                                                                                                                                 (see “Materials
                                                                                                                                                                                         48 h to deplete
                                                                                                                                                                                                                      and Meth-
                                                                                                                                                                                                              ERs of endog-
                                                                                                                                  enously     bound steroid.         ERs were subsequently         measured       in the super-
                                                                                                                                  natant fraction       of the cell homogenate          using a standard       ligand-bmnding
                                                                                                                                  assay.



  1                    ,1JI1T                                                    ULLul         :
                                                                                                                                                          Clone

                                                                                                                                                       Control
                                                                                                                                                         1V1
                                                                                                                                                                                                               ER (fmol/mg


                                                                                                                                                                                                                           238
                                                                                                                                                                                                                                   protein)




                                                                                                                                                           1v4                                                             831
                                                                                                                                                         1V5                                                               845
                                                                                                                                                       PKC-a
                                                                                                                                                         IA2                                                               989
                                                                                                                                                         IA3                                                               331
                                                                                                                                                         IA                                                                277
  (a1                                             _SS_                 #{149}
                                                                                                                                                       Control
  C,                                                                 :!                                                                                  JV1
                                                                                                                                                         JV4
                                                                                                                                                                                                                           266
                                                                                                                                                                                                                           582
                                                                                                                                                         JV5                                                               373
                                                                                                                                                       PKC-/31
                                                                                                                                                         JA2                                                               160
                                                                                          -                                                              JB1                                                               187
                                                                                                                                                         JD6                                                                69




 11                                                                                                                               were         less
                                                                                                                                  12). The influence
                                                                                                                                                          invasive            than
                                                                                                                                                                              of PKC-a
                                                                                                                                                                                        their        respective
                                                                                                                                                                                                    infection           was
                                                                                                                                                                                                                           control
                                                                                                                                                                                                                                 highly
                                                                                                                                                                                                                                           clones
                                                                                                                                                                                                                                              significant
                                                                                                                                                                                                                                                          (Fig.


Fig. 4. Representative      Western                         immunoblots      of wild type, vector only-                           (P     =     0.0001),            whereas            that      of PKC-1             infection    was of bor-
infected (1V2), and PKC-a-infected                           (IA2 and IA6) MCF-7 cells. Experimental                              derline           statistical             significance            (P =          0.0552),     primarily as a
conditions  are identical  to those                        described    in the legend to Fig. 3. Again,
                                                                                                                                  result           of the          poorly        invasive             nature        of one           of the         control
Westem         immunoblots                were repeated              numerous           times to include addi-
tional clones with identical  results. The a isoform was barely detectable  in                                                    clones           (JV2). Finally,              we evaluated                   the influence           of PKC over-
control cells (Wild Type and 1V2), whereas       the /3 isoform was never de-                                                     expression    on in vivo growth   in nude mice.                                                  Table 2 shows
tected. The 0 isoform     was not expressed    by any of our clones (data not
shown).                                                                                                                           that tumonigenicity     was markedly     reduced                                                 as a result   of
                                                                                                                                  PKC-a             infection.          Two          of the overexpressing                         clones        (IA3 and
                                                                                                                                  IA)         manifested                an       -75%               reduction            in tumor             formation,
                                                                                                                                  whereas             one         (IA5) did not form                   any tumor              at all in nude           mice
membrane                with        characteristic                   microvilli           (Fig.          1 0). There-
                                                                                                                                  (Table           1). Fig.          13 shows                the     overall       tumor          growth          in nude
fore,      it appears              that     as a result              of the        increase              in cell     size,
                                                                                                                                  mice of control                      (1V2 and lV4) and PKC-a-infected                                         (IA3, IA5,
some         folding         may           have          occurred,              which         created         the      for-
                                                                                                                                  and IAJ MCF-7                        cells. In addition to the reduction                                     in tumor-
mation  of holes in the cells on sectioning    of the cell block.
Fig. 10 also shows    the presence    of numerous    lysosomes,                                                                   igenicity,             the       growth            rate     of established                   tumors          was        also

which        were          more           prominent             in PKC-overexpressing                                cells        markedly   reduced                           (P < 0.0001)     by PKC overexpression.
                                                                                                                                  Table 3 summarizes                            the levels of PKC activity and ER meas-
than       in control             cells.       To determine                     whether            the     observed
morphological                     changes                were      indicative            of a more                 differ-        ured        in the tumors                 growing           in nude           mice.      The results           indicate
                                                                                                                                  that       most         tumors         derived             from         PKC-a-infected               MCF-7           cells
entiated           phenotype                 induced            by PKC              overexpression,                     we
                                                                                                                                  retained            a moderately                higher            level of enzymatic                activity        com-
evaluated  lipid                  droplet   formation      in our clones                            using the oil
red 0 staining                     techniques.        Overall,   we did                             not observe                   pared            with           mammary                   cancers            originating            from          control
striking         differences                   between               control            and        PKC-overex-
                                                                                                                                  clones.           Most          noticeably,            PKC-overexpressing                          tumors        exhib-
                                                                                                                                  ited       a drastic         reduction              in cytosolic   ER content                       (Table      3). This
pressing           cells       either        in the          absence              or in the          presence                of
PMA        (data       not        shown).                                                                                         could at least                  in part account   for our in vivo findings,   because
                                                                                                                                  tumorigenicity                    and growth    of MCF-7     breast    cancer   cells in
     Next,      we         assessed               the       anchorage-independent                              growth
                                                                                                                                  nude         mice        require            estrogen             supplementation.
properties             of our         cells.         As      can      be seen             in Fig.          1 1 , basal
clonogenicity      in soft agar was not affected                                              by PKC overex-
pression.     However,    in the presence of PMA,                                             both PKC-a and                      Discussion
-13 -infected    cells formed      significantly     fewer colonies    than                                                       The         major         objective            of our             experiments               was         to elucidate
their respective      control  clones.                                                                                            isoenzyme-specific                       functions  of PKC                        in breast    cancer                biol-
    Of most importance,       we observed        that induction   of PKC-a                                                        ogy. To address                      this issue, we infected                        hormone-responsive
and -13 ovenexpression      resulted  in a less aggressive                                                         breast         MCF-7             breast         cancer        cells        with        cDNAs         coding       for two         of the
cancer  phenotype.     Using an in vitro Matrigel  invasion                                                        assay,         conventional                    isoforms,           a and           These
                                                                                                                                                                                                     f3    .  isoenzymes     are ei-
we      showed             that      PKC-overexpressing                             breast          cancer           cells        then       minimally              expressed                (a) or not expressed     at all (fri) by
I 190   PKC Isoenzymes                  and Breast Cancer Phenotype




                                                                                                0.5% Serum
                         A                                                                                                           B


                                                                                                                          U)

                                 n=5                                                                               2                     n=2
         E                                                                                                         E
         C                                                                                                         C


                                                                                                                   8
                 U)                                                                                                       U,




                                 0             2               4           6                8                                            0        2           4          6            8

                                                           Days                                                                                           Days




                                                                                                                                                                                                       Fig. 5. Basal         anchorage-de-
                                                                                                 1% Serum                                                                                              pendent      growth  of vector    only-
                                                                                                                                                                                                       transfected         and PKC-trans-
                         A                                                                                                           B
                                                                                                                                                                                                       fected     #{149}
                                                                                                                                                                                                                     MCF-7    breast cancer
                                                                                                                                                                                                       cells in the presence      of the mdi-
                                                                                                                                                                                                       cated     serum concentrations.       A,
                                                                                                                          U)


         E
                                 n=3                                       4fl                                     2
                                                                                                                   E
                                                                                                                                         n=2                                                           13i -transfected
                                                                                                                                                                                                       fected cells.
                                                                                                                                                                                                                          cells; B, a-trans-
                                                                                                                                                                                                                        See “Materials       and
         C                                                                                                         C
                                                                                                                                                                                                       Methods”     for experimental          de-
                                                                                                                   8                                                                                   tails. Columns,    means; bars, SD;
                                                                                                                                                                                                       n, number       of replicate     experi-
                 U)                                                                                                       U)
                                                                                                                                                                                                       ments performed. In each exper-
                                                                                                                                                                                                       iment, two vector only- and three
                                 0             2               4           6                8                                                     2           4          6                             to     six  PKC-infected         MCF-7
                                                                                                                                                                                                       clones      were       simultaneously
                                                           Days                                                                                           Days                                         tested in duplicate.




                                                                                    iLl-i
                                                                                                5% Serum
                         A                                                                                                           B

                                                                                   r
         E
                                                                                                                                         n=3
         C



                 U)

                                                                                       .4                                 _
                                 0             2               4           6                8                                            0        2           4          6            8

                                                           Days                                                                                           Days




        our wild-type                  MCF-7          cells.        We were,         indeed,            quite     success-                     report,     Bryant       et     a!. (1 0) implicated               the     up-regulation            of
        ful in generating                     several        stable       clones            of PKC-overexpress-                                PKC-O as the factor              responsible           for acquisition         of an aggres-
        ing cells,         which         have        maintained            their       high       enzymatic             activity               sive behavior   by MCF-7  cells transfected   with the a isoen-
        for more             than 2 years.     Our data show    significant                                              cross-                zyme. In contrast,  we observed     that none of the other iso-
        regulation             among different    PKC isoenzymes.        We                                            demon-                  forms    were         affected    in our              cells following           induction       of
        strate        for the first            time        that      induction         of PKC-f3           overexpres-                         ovenexpression            of PKC-a   and              p. In particular,         we have        not
        sion is associated        with                      up-regulation               of the a isoform.   The                                detected    any expression    of the 0 isoform                        in any of our clones.
        reverse  cross-regulation                            was likewise               observed,   as also re-                                This different   pattern   of cross-regulation                          is likely to explain
        cently         reported          by Ways             et a!. (8). Our data,                  however,             signif-               the drastic        difference         in biological        behavior        exhibited       by our
        icantly         differ         from        those           of Ways         et a!. (8) with                regard           to          PKC-a-        and --ovenexpressing                     MCF-7        cells compared           with
        cross-regulation                   of other isoforms,    which   is likely to have                                                     those     engineered            by Ways       et aL (8). In the aggregate,                 these
        major biological                  consequences.     These authors     reported that                                                    results    emphasize            the    important        point     that     a specific      breast
        induction            of PKC-a               overexpression                 in MCF-7              breast         cancer                 cancer    phenotype    is not likely to be induced                          by a single      PKC
        cells         led to down-regulation                            of the              and     i     isoforms             and             isoenzyme      but by the coordinate     expression                          of multiple      iso-
        up-regulation                  of the 0 isoform                 (8, 1 0). In a recent                   preliminary                    forms.
                                                                                                                                                                                                   Cell Growth              & Differentiation            1191




                             A.                                                                                          clones,          was       either          unchanged                 under        basal           conditions            or
                    13.0                                                                                                 actually         reduced            in the         presence            of PMA          (Fig.       1 1).
                                                                                                                             Overall,         the most               significant            finding        of our experiments
                    12.5
             a.                                                                                                          was       that      induction                           and -13 in
                                                                                                                                                                   of overexpression                      of PKC-a
         .                                                                                                               MCF-7 breast cancer cells conferred      a less aggressive    phe-
                    12.0
         z                                                                                                               notype. This was characterized     by reduced in vitm invasive-
                                                                                                                         ness (Fig. 12) and, more importantly,      decreased    tumonige-
                    11.5
                                                                                                                         nicity, as well as growth of established   tumors in nude mice
         !                                                                                                               (Table      2 and Fig. 1 3). These                         results       are again           at variance            with
                    10.5
                                                                                                                         those       of Ways           et a!. (8), who observed                         accelerated                 growth       in
                                                                                                                         nude       mice        with       their      PKC-a-ovenexpressing                              cells.       Of note,
                    10.0                                                                                                 our PKC-a-infected                         cells generally               retained            a higher         level of
                               1           2              3            4           5              6          7
                                                                                                                         enzymatic              activity       when              growing          in nude             mice         compared
                                                                Time       (day)                                         with control tumors (Table 3). Whether such a modest                                                           degree
                             B.                                                                                          of PKC overexpression        was directly     responsible                                                     for the
                    12.5                                                                                                 marked attenuation    of tumor growth     in vivo remains                                                    unclean,
                                                                                                                         because     the much higher degree of overexpression                                                               ob-
             a.     12.0                                                                                                 served in vitro failed to influence cell proliferation.                                                      Alterna-
         .                                                                                                               tively,     it is possible                that     our PKC           measurements,                        performed
                    11.5                                                                                                 in total     cell      homogenates,                     may       have       failed     to detect            a much
                                                                                                                         greater difference     in membrane-associated        active PKC be-
       U                                                                                                                 tween the control and PKC-a-infected          cells. We believe that
         !          11.0                                                                                                 our in vivo findings can probably        best be explained      by the
                    10.5                                                                                                 profound    down-regulation     of ER levels observed       in the tu-
                                                                                                                         mors derived from PKC-a-infected          MCF-7 cells (Table 3). As
                    10.0                                                                                                 mentioned     above, PKC is well known to down-regulate             ER
                               1           2             3             4           5              6          7           expression              in these            cells        (9), although                the     specific         isoen-
                                                                Time (day)                                               zymes        involved             have            not      been       defined.              The     reason          why
                                                                                                                         induction           of PKC            overexpression                      induced             such         a drastic
Fig. 6. TGF-a sensitivity       of vector only- and PKC-transfected        MCF-7                                         down-regulation       of ER in vivo but not in vitro in our expeni-
cells with either the     (A) or a (B) isoform.      See “Materials and Methods”
for expenrnental details. Data represent         growth   curves by linear regres-
                                                                                                                         ments    is unknown.      In any event, in the presence of minimal
sion derived    from two replicate       experiments     per panel (each done in                                         amounts     of ER, MCF-7 cells would not be expected                 to be
duplicate). In each experiment,      two control and three PKC-overexpressing                                            very tumongenic        in nude mice, unless loss of ER is a corn-
clones were simultaneously        tested. - - -, vector without TGF-a; 0- - -0,
vector            with TGF-a;         -,   PKC without            TGF-a;           -U,      PKC with TGF-a.              ponent of their progression         to a hormone-independent           and
Statistical         analysis       revealed a significant(P < 0.0001)TGF-a    effect but not                             aggressive     phenotype,      as in the study reported by Ways et
a significant              difference in TGF-a sensitivity between      control and PKC-
overexpressing                 clones.
                                                                                                                         a!. (8). In contrast     to our findings,    these  investigators      ob-
                                                                                                                         served a major down-regulation            of ER expression        and ER-
                                                                                                                         mediated            transcription                  in PKC-a-transfected                             MCF-7           cells
    A major                 finding        of our study                was       the      observation             that   grown in vitro. More importantly,  they found that PKC-over-
PKC-overexpressing                             cells     exhibited          much       more       striking       mor-    expressing    MCF-7 cells were able to form large tumors     in
phological                 changes                    cellular flat-
                                            in response                to PMA                 ,                          nude mice in the absence of estrogen supplementation    of the
tening and vacuolization)    than control clones (Figs. 8 and 9).                                                        host. This observation                           clearly      shows       that,        in their expenimen-
Because PMA has been postulated          to have a differentiating                                                       tal system, loss of ER reflected       acquisition      of autonomous
effect in MCF-7 cells (1 1-13), we attempted      to document        a                                                   growth no longer dependent         on the estrogenic       milieu of the
more differentiated   state by assessing lipid droplet formation                                                         host. Cleanly, this did not occur in our study, because in the
using  the oil red 0 staining     technique.    We were unable,                                                          absence of estrogen pellets, neither control (to be expected)
however,               to detect            consistent             differences             in lipid      vacuole         nor PKC-a-infected       MCF-7 cells grew at all in nude mice.
accumulation                       between             control
                                     and PKC-overexpressing                                                                  Overall, basal anchorage-dependent           growth    did not sub-
cells either in the absence  or in the presence    of PMA (data                                                          stantially   differ between   control and either PKC-a on -p1                                                               -


not shown).    It remains unknown     whether    we would have                                                           infected MCF-7 cells when tested at different serum concen-
been able to demonstrate     a more differentiated    phenotype                                                          trations (Fig. 5) or in serum-free     media (data not shown).         In
with         the     use of other              markers           of differentiation.                                     addition,         sensitivity             to the proliferative                  action            of TGF-a          was
   These morphological     changes    are quite in contrast     with                                                     not influenced       by PKC overexpression       (Fig. 6). A feedback
those reported by Ways et a!. (8) in their PKC-a-transfected                                                             loop between EGF (and the EGF homologue                    TGF-a)    and
MCF-7 cells. These investigators      observed    the loss of epi-                                                       PKC has been postulated           in a variety of cell types (14, 15).
thelioid appearance,   indicative  of a less differentiated     phe-                                                     Activation      of PKC by diacylglycerol       generated    via ligand-
notype, along with an increase in clonogenicity       conferred   by                                                     induced       EGF receptor    phosphorylation     may down-regulate
PKC-a               transfection.              In contrast,                the   clonogenicity               of our      high-affinity     EGF binding, thus reducing     EGF signaling.    In our
PKC-overexpnessing                              cells,        compared             with    that       of control         experiments,               we       did      not        specifically           address              the     issue      of
1192   PKC Isoenzymes            and Breast    Cancer      Phenotype




             A.

             t0




             U)




        w




             C.)




                                                                                                                                          Fig. 7. Effect          of PMA administration            on the
                                                                                                                                          growth      of vector only-transfected        MCF-7 cells or
             C”
                                                                                                                                          CellStranSfeCted         withthe      4)ora    (B) isoenzyme.
                                                                                                                                          See “Materials        and MethOds”      for experimental      de-
                                         2                 3                   4             5          6                  7              tails Data represent           growth   curves by linear re-
                                                                        TNE    (DAY)                                                      gression.     In each experiment,       two vectoronly-      and
                                                                                                                                          three PKC-infected            clones   were simultaneously
                                                                                                                                          tested in duplicate. Statistical analysis revealed no
                                                                                                                                          significant     difference     in PPM sensitivity      between
             B.                                                                                                                           control     and PKC-overexpressing            cells. -,     PKC
              S
                                                                                                                                          without     PMA; L-L         PKC with 10 n PMA +-+,
                                                                                                                                          PKC with 100 nM PMA -, control without PMA;
                                                                                                                                          t-Li,     control with 10 nM PMA +“-+,             control with
                                                                                                                                          100 nM PMA.




                                                                        -.




              C.)




              CU




                                     2                          4                        6                    8
                                                                        TIME (DAYS)




       “transmodulation”        of EGF receptors      by PKC by performing                                  serving such PKC-mediated        effects will be important to iden-
       labeled EGF-binding         studies in our cells. However,        the ob-                            tify critical regulatory  steps within the signal transduction
       served failure of PKC overexpression            to alter cellular sensi-                             pathway      that may be targeted for therapeutic     intervention.
       tivity to the proliferative    effect of TGF-a indicates that, in our
       system,      such transmodulation       did not occur, at least in a                                 Materials          and    Methods
       biologically     relevant manner.                                                                    cell   Lines   and   Cultur     Conditions
           We report the novel observation          that induction     of PKC-a                             Wild-type   MCF-7 breast cancer cells (kindly provided by Dr. V. C. Jordan,
       and -13 overexpression          in MCF-7    breast   cancer cells pro-                               University  ofWisconsmn,        Madison,     WI; and Dr. John Isaacs, Johns Hopkins
       motes a more benign phenotype.             This finding indicates that                               University,  Baltirnore,    MD)ar,d      the transfected  clones were propagated         in
                                                                                                            75-cm2 flasks in Richter’s improved MEM (Life Technologies,                Inc., Grand
       it is possible           to influence      breast       cancer        behavior   in a favor-
                                                                                                            Island, NY) containing phenol red and 5% fetal bovine serum In a humid-
       able fashion             by modifying      the cellular          expression      of specific         rfied atmosphere                                                  In
                                                                                                                                   of 95% aIr and 5% CO2 at 37#{176}C. selected              experi-
       PKC          isoforms.     Investigation      of the cellular           mechanisms        sub-       ments, the culture       conditions       were stepped    down to steroid-depleted,
                                                                                                                                                                            Cell Growth         & Differentiation   1193




              OnM
                                                        -




                . .;   N
                                                                                                                                                F



                                                                                                                               %        .




                                                                                                                         _4


                                                                                                           ,    -          .                ... .




                                                                                                                                                      .;
                                                                                                       ‘                                            I..
                                                                                                                    .-              ,       :        .




              mM
                                                                                                                                                                                           ;;




                                                                                 .




             mOnM




             moonM




                                                                                                                          ‘p.,.?.




Fig. 8. Morphology     of vector only-infected     (left) and   PKC-a-infected       (right)   MCF-7   breast            cancer     cells                in the absence   and   presence        of the indicated
concentrations   of PMA administered      for 4 days.
1 194   PKC lsoenzymes            and Breast       Cancer       Phenotype




                   OnM
                         --:i.:. .             .      -.    -




                   mnM




                    lOnM




                    .,

                     .        .

                         ‘,


                    lOOnM
                                                                                                    ..                    ‘a$,
                                                                     ...             -                                       .,“.,
                                                                    1’                                   “
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                                                                              ,.,
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                                                                                                                      ‘              “




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                                                                                                                                                          -              .       -   -.   -.




        Fig. 9. Morphology   of vector only-infected     (left) and                                          PKC-f31-mnfected                (right)   MCF-7   breast   cancer       cells     in the absence   and presence   of the indicated
        concentrations  of PMA administered      for 4 days.
                                                                                                                                                                                  Cell Growth       & Differentiation   1195




                                                                                                                   A.
                                                                                                                  600-



                                                                                                                  500-



                                                                                                          .       400-
                                                                                                              C

                                                                             ‘,...
                                                                                                              0                    -I--
                                                                                                                                                                               -I-
                                                                                     :t’                  2
                                                                                                          z
                                                                                                              E
                                                                                                                  200



                                                                                                                  100-


                               ,:                                                                                       0-
                                                                                                                                         6             r-:-i                         18
                                                                                                                                    t          0ontr                          I       pc   .   B, transtected
                                                                                                                  PMA                    0                     lOOnM                  0                    lOOnM
                .-                                                                                                                                                     Treatment



Fig. 10. Electron      microscopic   appearance      of the cellular  vacuoles    ob-                              B.

served      on light microscopy.   This representative      picture  is taken from
                                                                                                                  600
PKC-a-infected       MCF-7 cells treated with 10 n PMA. Arrows,           lysosomal
structures.
                                                                                                                  500-                                                         ---




                                                                                                          .400-
serum-containing            media    or to serum-free     media,     as indicated       in “Results”
                                                                                                              0
and figure legends. Clonogenicity               in soft agar was assessed as previously                   8
published           by us (16).                                                                                   300-
                                                                                                          2
                                                                                                              E
Plasmids        and Transfection           Technique                                                      z       200-

The full-length,         2.6-kb cDNA encoding  PKC-f31 , cloned into the EcoRl site



                                                                                                                                                                                                   iTL
of the retroviral         expression vector pMW (1 7), was kindly provided   by Dr.                               100-
B. I. Weinstein            (Columbia     University,    New York, NY). The full-length,
3.0-kb      PKC-a       cDNA      also cloned   into the EcoRI       site of the pMV7           vector                  0__             6                        3                   15
was a generous              gift from     Dr. Curtis    L Ashandel         (Purdue         University,
Lafayette,   IN). PA31 7 cells were transfected         with these constructs    or the
                                                                                                                               -    :         contra;                   I                      at;nsfected-J
                                                                                                                  PMA                    0                     lOOnM                  0                    lOOnM
vector only using a modified        calcium   phosphate      method  (18), as recently                                                                                 Treatment
reported    by us (19), and the culture medium            was used to infect MCF-7
cells. MCF-7 cells provided by Dr. Isaacs were infected with the PKC-a                                   Fig. 1 1 . Clonogenicity        in soft agar of control and PKC-overexpressing
cDNA, whereas those provided by Dr. Jordan were infected with the (.3                                    MCF-7 cells in the absence          and presence   of PMA. A, /3 - transfected     cells;
isoform.   G41 8-resistant   colonies     were isolated, expanded,     and tested for                    B, a-transfected       cells. In each experiment,     two vector only- and three to
PKC activity. Approximately               50% of the clones analyzed exhibited                    PKC    six PKC-infected        clones were simultaneously       tested.   Numbers     of deter-
overexpression,      which has remained                 stable     when   tested       on repeated       minations     are indicated     in the columns.   Columns,     means; bars, SE. Sen-
occasions     for more than 2 years.                                                                     sitivity to PMA was significantly        greater (P < 0.05) in PKC-overexpressing
                                                                                                         than in control cells.

PKC      Activity
Near-confluent         MCF-7 cells were washed twice with ice-cold                Ca2 - and              total counts of the sample (minus the background          in the absence of the
Mg2 -free Dulbecco’s             PBS, followed     by two more washes with ice-cold                      substrate)  by the specific activity of the reaction mixture and the mm (e.g.,
equilibration      buffer [20 mM Tris-HCI (pH 7.5)] containing            2 m EDTA, 2 m                  1 5 mm) of the incubation    time.
EGTA, 250 mM sucrose,                 2 m.i DTT, 1 m         benzamidine,    and protease
inhibitors    (50 jg/ml      leupeptmn, 2 g/ml       aprotinin,   0.2 m AEBSF, and 10
g/ml       pepstatin     A). The cells were harvested           by scraping   from culture               Western              Blot Analysis
dishes into ice-cold           equilibration  buffer containing        1 % NP4O and then                 For Westem                blot analysis, cells were grown to 80-90%    confluency in a
disrupted      by 40 strokes         with a Dounce     homogenizer.       The homogenate                 1 00-mm Petri     dish and lysed with 500 pi lysis buffer [0.25 M Tris-HCI             (pH
was extracted for 1 h at 4”C, centrifuged at 100,000 x g for 60 mm, and                                  6.8), 5% SDS,      and 2 mM /3-mercaptoethanol].          The lysate was boiled for 5
passed through a DE 52 column to partially purify PKC. Twenty-five                       .tl of          mm, frozen on     dry ice, stored at -20#{176}C, and used within 1 week. Typically,
the eluate containing           30 g protein were chosen,            because    this volume              20 pi of each    fraction,   diluted 1:1.5 with 4x SDS sample buffer [150 mm
was found to be in the middle of the linear range of the assay. The reaction                             Tris-HCI (pH 6.8), 10 mM 2-mercaptoethanol,              2% SDS, and 20% glycerol],
was performed at pH 7.5 in Tris buffer containing 1 m calcium acetate,                                   were loaded on each lane of a 12% SDS-polyacrylamide                  gel, resolved      by
6.7 m a-phosphatidyl-L-serine,     3.2     PMA, 2.5 m DTT, 15 m mag-                                     electrophoresis,     and electrophoretically      blotted onto polyvinylidene       diflu-
nesium acetate,     50 M ATP, 10 pCVml [y2PJATP,        and 75 M histone IllS.                           oride membranes.         The Western      blots were probed      with antibodies     spe-
The incubation     was carried out for 1 5 mm at 25”C and terminated      by the                         cific for the different    isoforms   in the absence      and presence     of a 2.5-fold
addition  of a trichioroacetic  acid quenching   reagent (Amersham     kit RPN                           excess of the corresponding peptide. Monoclonal antibodies against the
77). The reaction            mixture    was spotted       onto phosphocellulose                binding   a,    a, and   isoforms were obtained from Transduction   Laboratories
disks, washed      in 5% (v/v) acetic acid twice fo 10 mm, and counted       in a                        (Lexington,     KY), and that against   the /3 isoform   was from SeigaKagu
scintillation counter.    PKC activity   was expressed    as pmoVmin      of 32P                         America     (Rockville, MD). The anti-is polyclonal    antibody  was obtained
incorporated    into histone IllS. This value was calculated   by dividing    the                        from Santa Cruz Biologicals     (Santa Cruz, CA).
1196   PKC Isoenzymes                 and Breast Cancer Phenotype




                40

                35

           30
                                                                                                                                                       Fig. 12.In vitro invasion of MCF-7 cells infected
                                                                                                                                                   with PKC-a (IA3, IA5 and IA6) and -/31 (JA2, JA5, and
           20                                                                                                                                      JC3). IV2, IV4, andlV5, and JV2, JV4, and JV, control
                                                                                                                                                   clones (infected with the vector only) for each set of
                15                                                                                                                                 overexpressing clones, respectively. Data represent
                                                                                                                                                   means.    Bars, SE. The numbers of experiments
           U    10
                                                                                                                                                   (each done in duplicate)     are shown in the columns.
                  5                                                                                                                                    See    text    for statistical        analysis.




                                                                                    Clone



       Table 2 Tumorigenesis in nude mice of control                            and PKC-a-infected
       MCF-7 breast cancer cells
           Ovariectomized athymic mice (15 per group) were injected with 5 x 106
       cells in two mammary      fat pads per mouse. The number of tumors that
       developed     versus the number of sites at risk (two per mouse) is shown.                                                                                                                                                 ‘V5

                 Clone                  No. of tumors                 No. of sites     at risk          %

                Control
                  1V2                            25                            30                      83
                  1V4                            25                            28                      89
                PKC-a
                  IA3
                     IA
                                                  6
                                                  0
                                                                               30
                                                                               30
                                                                                                       20
                                                                                                        0
                                                                                                                  I
                     IA                           8                            30                      27
       a   One mouse in this group                    died.




       ER Measurements
       For determination     of ERs, logarithmically      growing    breast cancer cells were
       plated in 150-cm2 flasks containing regular growth medium. At near
       confluency,     the cells were cultured       for an additional     48 h under steroid-
       free conditions (20) to deplete ERs of endogenously bound steroid. The
       cells were then harvested by brief trypsinization, washed once with PBS,
       resuspended in Tris buffer, sonicated for 20 s, and ultracentrifuged    for 30
       mm at 1 00,000 x g. The ERs were measured in the supematant          fraction
       using a standard  ligand-binding   assay (21).


       Eiq,.erlmental              Protocols                                                                                           5          10          15       20     25        30        35      40       45        50         55
       Control and PKC-overexpressing    MCF-7 cells were compared    with regard
                                                                                                                                                                                   Days
       to the following parameters.
          Proliferative ACtIVIty and Morphological   Features in Uquid culture.
       For evaluation of basal growth, the cells were plated in duplicate at a                                   Fig.   13.                in nude mice of control (1V4 and 1V5) and PKC-
                                                                                                                                In vivo growth
       density of 6 x io cells/35-mm      dish in regular growth medium containing                               a-infected (IA3, IA5, and IA6) MCF-7 cells. Ovariectomized  athymic mice
       5% serum. Twenty-four h later, the medium was replaced with either the                                    (15 per group) were injected with 5 x 106 cells resuspended      in 0.1 ml
                                                                                                                 medium in two mammary fat pads per mouse. The mice were also im-
       same or medium containing lower concentrations          of serum (0.5 and 1 %).
                                                                                                                 planted with 60-day, 0.72-mg E2 pellets. In the absence of E2 supple-
       Medium changes occurred every 48 h. Duplicate dishes per cell line were                                   mentation,  neither control                    nor PKC-overexpressing                    MCF-7 cells formed
       harvested   at the times indicated, and the number of cells was counted                                   tumors (data not shown).                      Data represent    mean             tumor     volumes  ± SE. In
       using a Couiter counter. For assessment of the effects of PMA on prolif-                                  constructing this figure, sites that did not develop tumors were included
       eration, the cells were plated in duplicate at a density of 6 x 1 0” cells/                               as 0. When assessing the influence of PKC overexpression    on the growth
       35-mm              dish in regular      growth     medium     in the presence        of 1 0 and 1 00 n    of established cancers, only tumors that did develop were included in the
       PMA (Sigma Chemical                     Company,        St. Louis, MO) or the same concentra-             analysis.
       tions of an inactive             4a-phorbol        ester.   The media   were changed        every 48 h,
       and duplicate dishes were                      harvested and counted at the times indicated.
       For assessment of effects                        of PMA on cell morphology, the cells were
       plated at a density of 2                       x iO cells/100-mm        dish. The experimental            addition       of 10 ng/mI        TGF-a.            Subsequent         media      changes        occurred          every
       conditions were otherwise                      identical. In experiments testing TGF-a sensi-             48 h, and duplicate                   dishes were harvested                   and counted              at the times
       tivity, 4 x i0 cells/35-mm                     dish were plated in duplicate in regular growth            indicated.
       medium.             After   24 h, the culture    conditions     were stepped   down to serum-                 Electron  Microscopy.     Electron   microscopy was performed  by fixing
       free media supplemented                    with 5 mr-.i glutamine,   0.2 mg/100 ml transfemn,             the cells for 1 h in 2.5% (v/v) glutaraldehyde and 0.2 M sodium cacodylate
       0.1 mg/100 mlfibronectin,    0.4 g/100 ml fraction V BSA, and 20 m,i HEPES                                buffer (pH 7.3). Following postfixation in 1% osmium tetroxide and 1.5%
       buffer. Forty-eight h later, the media were changed with and without the                                  potassium        ferrocyanide,              cells were dehydrated              through        a graded      series      of
                                                                                                                                                                               Cell Growth           & Differentiation              1197




Table 3 PKC actMty and ER levels of mammary
                                                                                                     analysis      of colony formation               the square root of the colony
                                                                                                                                                       in soft      agar,
                                                                       tumors   derived   from
control and PKC-a-infected MCF-7 cells                                                               number       was      the    appropriate   Unear models including a random
                                                                                                                                                         scale.
                                                                                                     clone effect       were always used. In each analysis, we modeled the effects
              Tumor                         P(cnit)Y                       ER (fmoVmg)               of transfection (PKC versus vector transfection).  experiment,    treatment
                                                                                                     (for the PMA and TGF-a) and higher-order Interactions     of the factors on
       Control
                                                                                                     colony formation or cell growth. With regard to the studies in nude mice,
                      1                            38.7                           65                 we assessed the effects of PKC overexpression    on tumorigertesis    and on
                                                                                                     the growth of established tumors. We assessed statistical significance    by
                      2                         21.1                             688
                      3                         31.1                             187                 approximate F tests and reported two-sided P values. Models were fit
                                                                                                     using Proc         Mixed       In the      latest      release         of the    SAS       statistical        software
                      4                         20.8
                      5                         27.8                             226                 package.
                      6                         20.9                             784
          Mean ± SE                         26.7 ± 2.9
                                                                                                     References
        PKC-a-infected
                      I                            63.2                                              1. AzzI, A., Boecoboinik, D., and Hensey, C. The proteln kinase C family.
                      2                            36.9                                              Eur. J. Biochem., 208: 547-557,   1992.
                      3                            53.8                                              2. Hug, H., and Sarre, T. F. Protein kinase C Isoenzymes:                                           divergence            in
                      4                            22.9                                              signal transduction?  BiOChem. J. 291: 329-343,   1993.
                    5                              42.1                                              3. O’BrIen, C. A., and Ward, N. E Biology of the protein                                        kinase        C family.
                    6                              53.0
                                                                                                     Cancer Metastasis   Rev., 8: 199-214,  1989.
                    7                              51.1                           11
                    8                              34.1                           15                 4. Costa, S. D., Fabbro, D.. Reggazzi, R., Kung, W., and Eppenberger,                                                    U.
                    9                              39.3                         <10                  The cytoeoilc phorboid receptor correlates  with hormone  dependency                                                     in
                                                                                                     six mammary            carcinoma           cell     lines.     Biochem.         Biophys.        Res.       Commun.,
                   10                              46.8
                                                                                                     133: 814-822,           1985.
                   11                              512
                   12                              38.9                            19                5. Fabbro,         D., Kung, W., Roes, W., Regazzi,                             R., and Eppenberger,                     U.
            Mean      ±    SE               44.4     ±     3.1                                       Epidermal  growth factor                binding and proteln kinase C actMties in human
                                                                                                     breast cancer cell lines:               possible  quantitative relationship. Cancer Res.,
                                                                                                     46:   2720-2725,            1986.
                                                                                                     6. Disatnik,  M. H., Winnier,   A. A., Mochty-Rosen, D., and kteaga,    C. L
ethanol and embedded          in Spurrs embedding                   medium.  Sections <90 nm         Distinct responses   of proteln Fdnase C lsoenzymes to c-erbB-2 activation
were stained     with uranyl acetate      and lead               citrate and examined   with a       In SKBR-3    human breast carcinoma cells. Cell. Growth & Differ., 5: 873-
Philips 400 electron     microscope.                                                                 880, 1994.
    Upid Droplet     Formation.                   was assessed
                                     This parameter               by the oil red                     7. BIobe, G. C., Sachs, C. W., Wasiuddin, A. K., Fabbro, D., Stabel, S.,
0 stainIng       technique.       and PKC-overexpressing
                                  Control                       MCF-7 clones                         Wetsel,  W. C., Obeld, L M., Fine, R. L, and Hannun, V. k Selective
were    grown in the absence  and presence    of 10 nu PMA on glass cover-
                                                                                                     regulation      of expression           of protein           kinase    (PKC)    isoenzymes               in multidrug-
slips  to approximately 80% confluency. The cells were rinsed twice with
                                                                                                     resistant      MCF-7 cells. J. Bid. Chem., 268: 68-664,                                    1993.
PBS    and fixed for 10 mm in neutral buffered formalin. FOllOwIng fixation,
the   cells were immersed    h absolute    propylene   glycol  for 2 mm and                          8. Ways, D. K., Kukoly, C. A., deVente, J., Hooker, J. L, Bryant, W. 0.,
subsequently   stained  in oil red 0 for 1 h. Subsequently, the cells were                           Posekany, K. J., Fletcher, D. J., Cook, P. P., and Parker, P. J. MCF-7
placed Into 85% propylene glycol solution for 1 mm, rinsed with dH2O and                             breast cancer cells transfected with protein kinase C-a exhibit altered
counterstalned            in Mayer’s hematoxylin          solution for 2 mm. After immersion         expression  of other protein kinase C isoforms and display       a more
in ammonia         water (0.23% ammonium hydroxide in dH2O) for 30 s and                             aggressive  neoplastic   phenotype.   J. Clin. Invest., 95: 1906-1915,
furtherdnsing       with dH2O, the cells were mounted               onto SIIdeSWIth glycerol         1995.
gelatin.                                                                                             9. Martin,    M. B., Garcia-Morales,    P., Stoica, A., Harrison,  B. S., Pierce, M.,
    Clonogenlcity         in Soft Agar. Plating in soft agar was done as de-                         Katz D., Zhang,          S., Danlelsen, M., and Secede, M. Effects of 12-0-
scnbed      previously     (16). Cells were plated in triplicate at a density of 1 x                 tetradecanoylphorbol-13-acetate         on estrogen      receptor activity in MCF-7
 i0 ceIlsIS5-mm dish. The number of colonies(aggregates                         >50 cells) was       cells. J. Blol. Chem., 270: 25244-25251,           1995.
scored on day 15.                                                                                    10. Bryant, W. 0., Kukoly, C. A., Garris, T. 0., Cook, P. P., Parker, P. J.,
    In Vitro Invasiveness.           Transwell     cell Inserts werecoated        with Matrigel      Pettit, G. R., and Ways, D. K. Anchorage independent growth of breast
in 0.1 % BSA and MEM to form a thin continuous                         barrier on top of the         cancer   cells, Role for PKC-0 In inhibiting anoikis (Abstract P2-639).  In:
Insert. 3T3 conditioned          media was used as a chemoattractant.                 Cells (1 x     Program     of the 77th Annual Meeting of The Endocrine Society: June
10/mI) were added to duplicate                 wells. After 12 h of incubation          (optimal     14-17, 1995, Washington,      DC, p. 450. Bethesda, MD: The Endocrine
time experimentally          determhied       by us), the number of MCF-7 cells that                 Society,      1995.
transversed       through the Matrigel and onto the inserts was quantified                      by
                                                                                                     1 1. Darbon, J. M., Valette, A, and Bayard, F. Phorbol esters inhibit the
staining    the cells with Duff Quick (Baxter) and counting.
                                                                                                     proliferation of MCF-7 cells. BlOchem. Pharmocol., 3: 2683-2686,    1986.
    Growth       In Nude Mice.          Ovarlectomized         athymic    Ncr-mi     mice were
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9 for 30 mm. Aliquots ofsupematants                  were used for measurement            of PKC
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In all cases,   we first analyzed the data to determine an appropriate scale
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1195   PKC lsoenzymes     and Breast   Cancer   Phenotype




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