246 Radiation Oncology, Biology, Physics Volume 32, Supplement 1

1046

ALTERATIONS IN CYCLIN DI EXPRESSION" IN HUMAN PROSTATE CARCINOMA

James V. Tfieoli and Laura M. Gumbiner

Department of Radiation Oncology, Division of Tumor Biology and Biophysics, Fox Chase Cancer Center, 7701 Burholme Avenue, Philadelphia, Pennsylvania 19111

Purpose: We have recently demonstrated that the retinoblastoma gene (RB 1 ) exhibits reduced expression in some cases of human prostate carcinoma. Cyclin D1 mediates the phosphorylation and inactivation of Rb, through the cyclin-dependent kinase 4 (cdk4), allowing progression through G1/S. Cyclin D1 expression has also been shown to be positively regulated by Rb protein. The purpose of this investigation was to determine whether alterations in the level of cyclin D 1 expression occur in prostate cancer. Such alterations could theoretically lead to changes in Rb function and cell cycle progression.

Materials & Methods: We have utilized quantitative RNA/PCR to determine the level of cyclin D1 lxanscripts in human prostate tumor tissue. Total RNA was isolated from frozen tumor tissue and 1 ug was used for cDNA synthesis using random hexamers and reverse wanscriptase. 20% of the cDNA product was used for PCR amplification using either primers for cyclin DI or tor a control gene c-N-ras. Amplification products were separated on a 2% agarose gel, transferred to a Nytran membrane, and hybridized with digoxigenin-labeled oligonucleofide which recognizes the amplified DNA fragment. DNA fragments were visualized by chemiluminescence on X-ray film and quantitation performed using a scanning densitometer. Total RNA from the LNCa.P prostate carcinoma cell line was used as a positive control for cyclin D1 and c-N-ras expression in these experiments.

Results: We have analyzed 35 samples of RNA isolated from human prostate carcinomas of differing stage and grade using RNA/PCR. We judged a greater than 50% change in signal over the LNCaP control as significant. Our results demonstrate that none of the samples known to have diminished levels of RB 1 transcript show any alteration in cyclin D1 transcript levels. However, we did detect significant reductions in cyclin D1 transcript levels in two of the tumor samples. The tumors were both pathologic stage B, and one was a Gleason grade 6 while the other was a grade 7. While several of the tumors demonstrated minor fluctuations in signal intensity, they fell under the 50% significance level. We did not observe an increase in cyclin DI levels in any of the prostate tumor RNA samples analyzed.

Conclusion: The results of this study demonstrate that only 6% (2/35) of the prostate tumor samples exhibit an alteration in cyclin DI transcript levels and that in both cases a reduction was observed. The fact that none of the tumors which were known to have reduced RB1 transcript contained lowered cyclin D1 transcript levels is surprising since Rb protein is known to positively regulate cyclin D1 expression. Therefore, either the reduced Rb level in these tumors is sufficient to maintain c yclin D l transcript at normal levels, or other factors compensate for the loss of Rb protein. Studies from other laboratories have shown that the cyclin D1 gene is amplified and overexpressed in human hepatocellular carcinoma and in osteosarcoma cells. In hepatocellular carcinoma the overexpression can be as great as ten-fold and is associated with more aggressive disease. However, in our set of 35 prostate carcinomas, we find no evidence of cyclin D 1 overexpression at the transcript level. Thus, cyclin D1 overexpression, which could lead to accelerated progression through G I/S phase through phosphorylation of Rb, is not occaring in these tumors. Reduced cyclin D l levels could lead to a greater level of hypo-phosphorylated active Rb, keeping some cells in check at the G I/S phase boundry, and resulting in a reduced proliferative role for this sub-population of tumor ce i l s How this may effect clinical progression, or what its relation to the role of Rb and p53 mediated response to radiation induced DNA damage may be, is under investigation.

1 O47

P53 MODULATION OF RADIATION INDUCED GI ARRESTS AND INTRINSIC RADIATION SENSITIVITY

Su, Mei M.D. and Pardo. F . S . M . D

Lalx~ratory of Molecular/Tumor Radiation Biology, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114

Objective: Wild type p53 functions as a cell cycle control protein at the GI/S cell cycle interface. DNA damage following exposure to ionizing radiation result in increases in p53 expression concomitant with cell cycle arrest at the G1/S boundary.. We sought to investigate the relationship between p53 expression and cell cycle arrest in REC transfected with p53 expression vectors.

Methods: REC were transfected wit both mutant and wild type p53 expression vector by a calcium phosphate-based method. Transfected cellular populations, consisting of 3-6 clones isolated from the transfection studies, were used for subsequent analyses. Radiation survival assays measured clonogenic survival following exposure to 250 kVp x-rays under oxic conditions. The data were fitted to the linear quadratic model of cell survival, emphasizing D and SF2 as parameters. Expression of p53 protein was determined in transfected cellular populations both prior to and following doses of 0,2,5,10, and 15 Gy. Flow cytomemc techniques assessed radiation-induced cell cycle changes for up to 48 hours following irradiation, with particular emphasis on the kinetics of both the G1/S and G2/M cell cycle transitions.

Results: Cellular populations transfected with mutant p53 express levels of p53 approximately 10-fold higher than untransfected or mock-transfected counterparts. REC transfected with wild type p53 were more sensitive to ionizing radiation in vitro (2-tailed test, SF2,MID). REC lransfected with wild type p53, as well as those that are untransfected or mock-transfected, reveal dose dependent arrests at the G1/S interface concomitant with modest elevations in p53 protein production. Cells transfected with mutant p53 demonstrate a lack of arrest following irradiation without significant upregulation of overall p53 protein production. REC transfected with a human mutant p53 allele, reveal increases in resistance to ionizing radiation in vitro (p<.05, SF2,MID/.

Conclusion: Following exposure to ionizing radiation in vitro, mock-transfected untransfected, and wild type p53 transfected REC reveal a dose dependent GI arrest. Although all cell lines demonstrate a dose dependent G2 arrest, the extent of the arrests, and the distribution of cells in pa~icular phases of the cell cycle, are cell-type dependent, and specific to the particular transfected population of ceils. REC transfected with mutant p53 show no G 1 arrest, but a significant G2 arrest. Such cell cycle 'alterations, particularly regulation of "checkpoints," are critical determinants of cell survival following exposure to ionizing radiation in vitro.