and Progesterone Receptor Concentrations in Human Uterus Tissue

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					CLIN. CHEM. 29/6, 1070-1072        (1983)




Stability of Estrogen- and Progesterone-Receptor Concentrations in Human
Uterus Tissue
N. V. Zacharlah and Z. H. Chakmakjian

Homogeneous control specimens for estrogen receptor (ER)               small pieces, and quickly frozen in liquid nitrogen. These
and progesterone receptor (PR) assays were prepared from               tissues were transported on solid CO2 to the laboratory             and
freshly collected human uterus. After removal of the connec-                                   until
                                                                       stored at -70 #{176}C analyzed.
tive tissue, the specimen was washed with isotonic saline,                 Preparation     of cytosol. The frozen pieces of uterine tissue
cut into small pieces, quickly frozen in liquid nitrogen, and          were pulverized in a pulverizer            (Thermovac Div., Island
stored at -70 #{176}C analyzed. Cytosol prepared from this
                      until                                            Park, NY 11558) that had been precooled in liquid nitrogen.
specimen was lyophilized and stored at -70 #{176}C.   A single         The pulverized         powder was suspended in three to five
step of reconstitution, with glycerol (100 mL/L) in water, is          volumes of ice-cold       buffer(per liter: 10 mmol of Tris HC1, pH
sufficient to prepare a control. Two specimens prepared this           7.4, 1 mmol of EDTA, and 0.5 mmol of dithiothreitol).               The
way were found to be reasonably stable for 20 months (first            sample was then homogenized (Polytron homogenizer,
specimen, mean ± SD: ER = 22.1 ± 2.9 fmol/mg, PR =                     Brinkmann        Instruments,     Westbury, NY 11590). The tubes
136.5 ± 26.9 fmol/mg; second specimen: ER = 107.2 ± 11.7               were constantly         kept in crushed ice. Four 15-s bursts at
                                                                       approximately        1-mm intervals gave a consistent homoge-
fmol/mg, PR = 922 ± 71.6 fmol/mg). Another specimen,
                                                                       nate. The homogenate was transferred                to 25 x 90 mm
prepared similarly but not frozen in liquid nitrogen soon after
                                                                       polyallomer tubes (Beckman no. 326823) and centrifuged               for
collection,  was less stable; its ER and PR concentrations
                                                                       45 mm (105 000 x g). The temperature was maintained at
deteriorated    faster.                                                       throughout
                                                                       2#{176}C              the centrifugation.   The clear supernate was
                                                                       filtered through glass wool and stored at 0#{176}C.
   In recent years, the presence of estrogen receptors (ER)’               Protein    determination.      An aliquot   of the cytosol was
and progesterone receptors (PR) in cancerous breast tumors             serially diluted in the buffer and the protein concentration
has been shown to correlate     well with response to endocrine        was determined         spectrophotometrically     (9). First, the vol-
therapy (1-3). As a result, these receptors in all surgically          ume of cytosol was adjusted to approximately 2.5 g/L with
resected breast cancers are routinely measured at all major            the buffer and its protein content was determined                by the
institutions (4-6).                                                    method of Lowry et a!. (10). After making several dilutions
   The methods commonly        used for measurement of these           of the cytosol, to obtain more accurate determination                 of
receptors include: sucrose density-gradient     ultracentrifuga-       protein concentration,        the cytosol volume was then adjusted
tion, adsorption on dextran-coated charcoal, single satura-            to exactly 2 g/L in the buffer. A few milligrams             of sodium
tion-dose analysis (7), gel filtration, electrophoresis   (2), and     azide was added and 2-mL fractions were aliquoted into 10-
histochemical    methods (8).                                          mL bottles and stored at -70 #{176}C.
   We have been quantifying ER and PR in breast tumors                    From homogenization   to storage, every step in this 10-h
since 1976, using successively sucrose density-gradient          ul-   process was carried out without interruption.
tracentrifugation,   dextran-coated charcoal, and single satu-           The following day, frozen cytosol fractions were lyophi-
ration-dose analysis. Although these methods have been                 lized overnight. Approximately  50 samples could be lyophi-
well established, each needs a control specimen to validate            lized in a single batch. The bottles were then corked and
the ER and PR assays. Because no commercial control was                                   ready
                                                                       stored at -70 #{176}C, for use as a control material.
available, we looked into the possibility of preparing our               Reconstitution of control lyophilized powder. On the day of
own control material from freshly collected human uterus.              the experiment, the lyophilized control was reconstituted in
This article describes the preparation and stability of ER             2 mL of glycerol/water   (10/90 by vol), mixed, and left on ice
and PR in human uterus tissue.                                         until used as a control     in assays of both ER and PR.
MaterIals and Methods                                                  Methods     of Analysis
Preparation       of the Control                                          Sucrose density-gradient      method: Linear gradients of su-
                                                                       crose (100-300      g/L) were prepared the day before and
   Collection of the human    uterus.   Human uterus      from
women undergoing     routine hysterectomy   for uncontrolled           equilibrated at 4 #{176}C  overnight. Two 250-L       samples of
                                                                       cythsol were incubated with 1.0 pmol of radioactive estradiol
dysfunctional bleeding was obtained at surgery. Connective
tissue was removed by dissection and the freshly collected                                                           One
                                                                       ([2,4,6,7-3H]estradiol-1713) for 4 hat 4 #{176}C. of the samples
uterus was washed in cold isotonic saline, promptly cut into           was incubated with 100 pmol of diethylstilbestrol       (DES) for
                                                                       15 mm before the tritiated estradiol was added.
                                                                          Charcoal pellets used to separate the free from bound
  Weinberger   EndocrineLaboratory, Section of Endocrinology and       radiolabeled estradiol were prepared as follows: 1 mL of a
Metabolism,    Departments of Medicine and Pathology, Baylor Uni-      suspension containing 2.5 g of charcoal and 25 mg of
versity MedicalCenter, 3500 Gaston Ave., Dallas, TX 75246.
                                                                       dextran per liter of Tris HC1 buffer (10 mmol/L, pH 8.0) was
   Presented in part at the 34th AACC national meeting, August
                                                                       centrifuged at 1600 x g for 10 mm and the supernatant fluid
1982, Anaheim, CA.
                                                                       discarded. The 250 pL of cytosol was transferred onto the
   ‘Nonstandard abbreviations: ER, estrogen receptor; PR, proges-
terone receptor; DES, diethylstilbestrol; HUP, human uterus pow-                                                    for
                                                                       pellet and the mixture was incubated at 4#{176}C 20 mm.
der.                                                                   After centrifugation, 200 L of the clear supernate was
  Received Nov. 4, 1982; accepted March 7, 1983.                       layered onto the gradients. These gradients were centri-


1070   CLINICAL CHEMISTRY, Vol. 29, No. 6, 1983
ftiged for 18 h at 207 000 x g. Then 0.2-mL fractions                 were
collected with a gradient fractionator        and their radioactivity
was measured with use of modified Bray’s scintillation
cocktail.
  Dextran-coated  charcoal (DCC) assay: We incubated 200
                                                                              1
L   of cytosol (1-2 mg/mL of protein) with increasing
amounts of radiolabeled estradiol, ranging from 15 to 200
pmo!. We also incubated 200 .tL of cytosol with 100 pmol of
DES for determination of nonspecific binding, and 200 L of
buffer blanks. Al! samples were incubated at 4#{176}C 16 h,   for
then 500 L of charcoal suspension was added to all the
tubes and incubated for 20 mm at 4#{176}C. centrifuging
                                                 After                        Fig. 2. Sucrose density-gradientanalysis for PR (A) and ER (
                      for
the tubes at 4#{176}C10 mm, we then counted the radioactiv-                   y-axis,cpm; x-axis, traction numbercollected
ity of the supernate, using Bray’s scintillation cocktail. The
data were analyzed by the method of Scatchard (11).                                1200
    Single saturation-dose     method: In this assay a single dose
of tritiated estradiol was used to saturate the receptors. We
incubated 200 L of cytosol with 0.25 pmol or more of
tritiated   estradiol                     A parallel sample of 200
                        for 4 h at 4#{176}C.                                       1000
jiL of cytosol was incubated with 100 pmol of DES 15 mm
before adding the tritiated      estradiol. After adding 500 pL of                                                 SliP *2     PR   1-$
charcoal suspension and incubating for 20 mm at 4#{176}C,         we
centrifuged     the samples at 1600 x g for 10 mm and counted
the supernate for radioactivity,         using Bray’s scintillation
cocktail. This method is especially useful when the amount
of the tumor tissue for analysis is limited and when multiple                  E

receptor determinations are needed on a single specimen.
     Progesterone receptors were quantified            similarly  (3).
R5020 (17,21-dimethyl-19-nor-pregna-4,9-diene-3,10-one)                       0.

and radiolabelled R5020 obtained from New England Nucle-
ar (Boston, MA 02118) were used for the PR assays.
                                                                                   100
Results
   Stability of human uterus powder (HUP) was studied for                                                          SUP *1      PR..-.
                                                                                                                   HOP H2      ER**
ER and PR concentrations for several months. To analyze
ER and PR, the dextran-coated charcoal assay was our
routine procedure, but we used sucrose density-gradient
ultracentrifugation  and single saturation-dose methods pe-
riodically to check the accuracy of the system. Figure 1
shows a Scatchard plot analysis for ER in HUP #1, and


                                                                                                                   SUP *1      ERO-O
1.0
                                                                                    10
  .9                                                                                       2     4    6     P    10       12        14    16   18   10
                                                                                                                MON’FHS

  .8                                                                          Fig. 3. Stability of ER and PR in HUP #1 and HUP #2 during20
                                                                              months
  .7                .1




  .6                                                                          Figure 2 shows the results of sucrose       density-gradient
                                                                              analysis for both ER and PR. The Scatchard plot confirms
  .5                                                                          the presence of ER (Kd 4.9 x 10     mollL, Ka 2.03 x 1010 L/
                                                                              mol), and the sucrose density-gradient assay confirms the
  #{149}14                                                                    presence of both 8S and 4S fragments of estrogen receptors.
                                                                                  To prevent loss of receptor activity, the freshly obtained
  .3                                                                          samples were quickly frozen in liquid nitrogen, transported
                                                                              on solid CO2 to the laboratory, and pulverized at liquid
  .2                                                                          nitrogen temperatures. Homogenization was at 0 #{176}C,   ultra-
                                                                              centrifugation between 2 and 4#{176}C.  Scatchard plot analyses
  .1                                                                          on the lyophilized powders showed no loss in receptor
                                                                              activity. After storage of the lyophilized samples at -70 #{176}C
                                                                              freezer,  HUP #1 had an average ER of 22.1 (SD 2.9) fmoll
           5    10    15     20 25       30         35    0      45      50   mg over a period of 20 months (Figure 3). The average PR
Fig. 1. Scatchardanalysis of ER binding sites                                 value over the same period for HUP #1 was 136.5 (SD 26.9)
Recepto content (temtomoles) is represented on the x-axis and B/F on the y-   flnol/mg.   In another human uterus powder (HUP #2),
axis. K = 4.9 2<10_li moVL K = 2.03 x iO’#{176}
                                              L/mol                           prepared earlier, the average ER for 20 months for HUP #2

                                                                                               CLINICAL CHEMISTRY, Vol. 29, No. 6, 1983             1071
                                                                        require   homogenization of the control specimen before use
     J,20
                                                                        in the assay.
 110
                                                                           The method we describe for preparing a homogenous
                                                                        lyophilized specimen derived from human uterus requires
 lOC                                                                    only one simple step of reconstituting the lyophilized control
                                                                        specimen with water. The homogeneity of the receptors is
      91                                                                maintained for a considerable period-as long as 20 months
                                                                        in quickly frozen, freshly collected human uterine tissue.
      8c                                                                Using such controls would greatly improve the quality of
                                                                        estrogen and progesterone receptor assays.
      7C
                                                                          We thank the staff of the Endocrine Laboratory for their technical
      6c                                                                assistance, the staff of Surgery and Pathology Departments for
                                                                        collecting and preparing  the specimens, and Catherine Higgins for
      5C                                                                her excellent assistance during the preparation of this manuscript.

      l4
                                                                        References
81    3
                                                                   PR   1. Byar DP, Sears ME, McGuire WL. Relationship           between estro-
                                                                        gen receptor values and clinical data in predicting the response to
     21                                                                 endocrine therapy for patients with advanced breast cancer. Ear J
                                                                        Cancer 15, 299-309 (1979).
     11 ER                                                              2. McGuire WL, Pearson OH, Segaloff A. Predicting hormone
                                                                        responsiveness in human breast cancer. In Estrogen Receptors in
            ______________________________________                      Human Breast Cancer, WL McGuire, PP Carbone, Eds., Raven
                      2                     6         8       10        Press, New York, NY, 1976, pp 17-30.
                                                 Months                 3. McGuire WL, Reynaud JP, Baulieu E. Progesterone          receptors-

Fig. 4. Stability of ER and PR in HUP #3                                an overview. In Progress in Cancer Research and Therapy, WL
                                                                        McGuire, JP Reynaud, Eds., Raven Press, New York, NY, 1977, pp
                                                                        1-8.
                                                                        4. Berchard P, Garola RE, Chaniness CC, McGuire WL. Measure-
was 107.2 (SD 11.7) and the PR was 922J. (SD 71.6) fInoIImg             ment of progesterone receptor in human breast cancer biopsies.
(Figure 3).                                                             Cancer Res 39, 1678-1682 (1979).
   HUP #3 was not frozen in liquid nitrogen soon after                  5. Witliff Mehta RG, Boyd PA, Coral JE. Steroid binding
                                                                                     JL,
collection, but the remaining  steps in preparing the lyophi-           proteins in the mammary gland and their clinical significance          in
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                                                                        advanced breast cancer. Breast 3, 29-31 (1977).
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                                                                        7. McGuire WL. Steroid receptors in human breast cancer. Cancer
uterus in liquid nitrogen.
                                                                        Res 38, 4289-4291 (1978).
Discussion                                                              8. Pertachuk LP, Gaetjens E, Carter AC, et al. An improved
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1072        CLINICAL CHEMISTRY, Vol. 29, No. 6, 1983

				
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