Mechanisms of G1 checkpoint loss in resected early stage non-small cell lung cancer.

Loss of the G1 checkpoint appears to be extremely common among virtually all neoplasms. A variety of genetic and epigenetic mechanisms have been demonstrated to play significant roles in this process. In a consecutive series of early stage non-small cell lung cancer (NSCLC), we have established the loss of expression of the G1 Cdk inhibitors p15INK4b) and p16INK4a by DNA methylation is very common (37%), and methylation of p16INK4a is tightly correlated with loss of expression of p16INK4a protein (P = 0.0018). Furthermore, methylation of p15INK4b and p16INK4a appear inversely correlated, although methylation of p15INK4b is an infrequent event in this cohort (4%). Methylation was detected in all stages of NSCLC equally, and did not correlate with survival in these patients. Evidence for methylation was more frequent in squamous cell carcinomas in comparison to other tumor histologies (P = 0.0156). In addition, over-expression of cyclin D1 was found to be tightly restricted (P = 0.0032) to those tumors that had retained wild-type expression of pRB, and did not correlate with methylation or expression of p16INK4a gene product. Although loss of p16INK4a function remains tightly correlated with pRB expression, loss of other regulatory elements in NSCLC such as p53 mutation and cyclin D1 over-expression appear independent of loss of the p16INK4a gene product.

Gene therapy of established mesothelioma xenografts with recombinant p16INK4a adenovirus.

The absence of expression of the p16INK4a gene product is observed in virtually all mesothelioma tumors and cell lines, whereas wild-type pRB expression is maintained. We have examined the potential therapeutic role of re-expressing the p16INK4a gene product in mice with established human mesothelioma xenografts. Experiments using Adp16 treatments in mesothelioma xenografts demonstrated prolonged survival and potential cure following treatment with p16INK4a-based gene therapy. These results demonstrate that p16INK4a gene transfer may play a therapeutic role in the treatment of mesothelioma.

Re-expression of p16INK4a in mesothelioma cells results in cell cycle arrest, cell death, tumor suppression and tumor regression.

Absence of expression of the p16IKN4a gene product is commonly observed in mesothelioma tumors and cell lines, while wild-type pRB expression is maintained. We have examined the biologic and potential therapeutic role of re-expressing p16INK4a gene product in mesothelioma cells and tumors. Following transduction with a p16INK4a expressing adenovirus (Adp16), over-expression of p16INK4a in mesothelioma cells resulted in cell cycle arrest, inhibition of pRB phosphorylation, diminished cell growth, and eventual death of the transduced cells. Expression of p16INK4a protein was accompanied by decreased expression of pRB as detected by immunoblot and immunohistochemistry. Experiments in mesothelioma xenografts demonstrated inhibition of tumor formation, tumor growth arrest and diminished tumor size and spread. p16INK4a gene product expression was also demonstrated in intraperitoneal xenografts of human mesothelioma cells. These results demonstrate that p16INK4a gene transfer may play a therapeutic role in the treatment of mesothelioma.

Human lung carcinomas express Fas ligand.

To reach a clinically detectable size, neoplasms must be able to suppress or evade a host immune response. Activated T cells may enter apoptosis in the presence of Fas ligand (FasL) (1), and tissue expression of FasL has been shown to contribute to immune privilege in the eye and testis (2, 3). We have demonstrated that all human lung carcinoma cell lines tested (16 of 16) express a Mr 38,000 protein consistent with FasL by immunoblotting, whereas the majority of resected tumors (23 of 28) show positive staining for FasL by immunohistochemistry. DNA sequencing of reverse transcription-PCR products from lung cancer cells and resected lung tumors confirms the presence of human FasL mRNA in these neoplastic tissues. Furthermore, lung carcinoma cells are capable of killing a Fas-sensitive human T cell line (Jurkat) in coculture experiments; this killing was inhibited by a recombinant form of the soluble portion of the Fas receptor (FasFc). FasL expression by neoplastic cells represents a potential mechanism for peripheral deletion of tumor-reactive T-cell clones.

Proliferative activity in benign and neoplastic prostatic epithelium.

The increasing availability of means for the early detection of prostatic cancer has brought under scrutiny the criteria used for prognosis and emphasized our limitations in understanding what determines the rate of progression in these cancers. The rate of cancer cell proliferation has been under intense investigation, which, however, has yielded conflicting results. In this study we evaluated the proliferative activity of benign and neoplastic prostatic epithelium, using various existing methodologies. We first analysed the variability introduced by the methodological approach and then attempted to demonstrate whether determination of the proliferative capacity had any clinical consequence that complemented the histological grading. Tissue samples from patients, 88 with cancer and 46 with benign prostatic pathology, were studied using in vitro bromodeoxyuridine (BrdU) incorporation as well as Ki67 and the proliferating cell nuclear antigen (PCNA) to estimate the proliferative activity. Increased proliferation was found consistently in inflammation and metaplasia, but not in hyperplasia. In contrast, cancers showed marked variability. Although average proliferation indices increased with grade, there was a wide scatter of values. Correlation was stronger with stage, but also depended on the methodology. Bromodeoxyuridine indices over 10 per mile had a positive predictive value of 79 per cent for cancers extending beyond the prostatic capsule and may prove particularly helpful for evaluating patients with grade 7 cancer. This observation is significant, since grade 7 cancers are the most frequent and the least predictable.