Table 1. Tumor gene mutations, protein expression, and in vitro growth properties of 17 primary cell cultures established from malignant glioma.
p53 Primary cell culture BMR-76 CRL-8 CNT-1 CDR-97 CPT-86 FCN-9 FLS-10 GSS-98 SPR-2 LTN-12 MRC-60 MSS-5 MTR-4 MZC-12 PMG-71 PST-7 PRZ-11
a b
Da
Gene statusb
Positive cells by IF (%)c 0 72 0 0 0 10 0 0 0 0 0 0 0 82 0 0 0
INK4A-ARF locus Gene status
EGR-1 Labelling indexd 50 481 107 218 183 42 46 420 132 136 65 173 135 589 35 251 115
Mdm2 Labelling indexe 0 50 24 85 28 52 42 10 27 45 44 42 20 30 41 21 26
Vimentin Labelling indexf + +/+ + + +/+ + + + +
GFAP Positive cells by IF (%) 0 31 0 0 23 68 77 0 0 0 0 0 82 70 0 0 0 Duplication time (hr) 38 66 96 42 280 52 64 40 69 100 87 36 72 52 87 122 96 Soft agarose cloning (%) 0 3.0 0 0 6 3.1 0.5 0 0.3 0.5 nd 1.7 0.1 6 nd 0 0
Plating efficiency High serum (10%) 35 40 40 0 45 30 12 0 20 40 40 17 46 11 25 15 80 Low serum (2,5%) 0 3 0 0 0 20 0 0 1.5 0 0 3 4 5 0 0 0
G G G G G A G A G G G G G G G A G
Homo wt Homo MUT 216 Homo wt Homo wt Homo wt Homo MUT 224 Homo wt Homo wt Homo wt Homo wt Homo wt Homo wt Homo wt Homo MUT 248 Homo wt Homo wt Homo wt
Homo wt Homo wt Homo wt Homo wt Homo wt Homo wt Homo DEL Homo DEL Homo wt Homo wt Homo wt Homo wt Homo wt Homo wt Homo wt Homo wt Homo wt
D, diagnosis defined as grade III anaplastic astrocytoma (A) or glioblastoma multiforme (G) When genotype is not normal (Homo wt), then the presence of point mutations (MUT) or allele deletions (DEL) is indicated. In the case of MSS-5, the mutation found at the p53 gene locus was detected only in the original bioptic sample, and not in the primary culture. c At least 300 cells from different fields were counted.
d
EGR-1 was detected by enhanced chemiluminescence after 30 minutes of film exposure. Values reported as labelling index corresponded to units of integrated O.D. measured by densitometric analysis of Western blot band scannings, and were normalized with respect to actin content of each sample. e Mdm2 fluorescence was detected in the total population of cells. The intensity of fluorescence was measured in four consecutive fields with a digital densitometer applied to the fluorescence microscope. The mean of derived OD values was calculated and reported as labelling index. Note that OD values may range between 0 and 255. f For Vimentin, the cell labelling intensity evaluated by immunocytochemistry is indicated by -, when vimentin is not expressed; +/-, when low to moderately expressed; +, when strongly expressed.