It is clear that in human cancer, including lung cancer, deregulation of cell cycle progression is a hallmark of neoplastic transformation and that genes involved in G1/S transition of the cell cycle are especially frequent targets for mutations. A cell-cycle related protein, the recently identified p16^INK4A gene product, plays an important role in the negative regulation of the kinase activity of cyclin dependent kinase(cdk) enzymes¹⁾. p16^INK4A gene is, thus, an important tumor suppressor gene and is also called MTS1(multiple tumor suppressive gene 1)^{2, 3)}. In various malignancies, reports of alterations in the p16^INK4A gene are much more common than those involving any other oncogene or tumor-suppressive gene ever identified^{2, 4, 5)} It was particularly noticeable that there was no p16 expression in more than 70% of cell lines examined in the case of non-small cell lung cancer. It is speculated that p16^INK4A gene could exert a key role in the development of non-small cell lung cancer⁶⁾. The purpose of this study was to investigate whether p16^INK4A gene is a suitable candidate for gene therapy in cases of non-small cell lung cancer. After the extraction of total RNA from normal fibroblast cell lines and subsequent reverse transcriptase and polymerase chain reaction, amplified p16^INK4A was subcloned into eukaryotic expression plasmid vector, pRC-CMV. Using lipofectin, the constructed pRC-CMV-p16 was transfected into NSCLC cell lines NCI-H441 and NCI-H157. After the extraction of proteins from cell lysates, the changes in G1 cell-cycle related proteins were investigated with Western blot analysis and immunoprecipitation and the tumor suppressive effect was observed by clonogenic assay.

A variety of evidence suggests that p16 is a tumor suppressor gene. p16^INK4A is located in chromosome 9p21, where deletions have frequently been found in carcinomas of lung, kidney, ovary, pancreas, breast, bladder, head and neck and esophagus and in several other tumors, such as glioma, melanoma, leukemia and osteogenic sarcoma^{2, 3, 7–9)}. In addition, homozygous deletions or mutations of the p16^INK4A gene are also frequently observed in cell lines from these tumors^{4, 10)}. Because deletion or mutation of p16^INK4A is significantly less frequent in primary cancer tissues than in cancer cell lines, it has been suspected, however, that p16^INK4A may not be a true tumor suppressor gene^{5, 11)}. In cases of non-small cell lung cancer, about 70% of cell lines do not express p16 protein⁶⁾, but mutation of the p16^INK4A gene is not frequent in primary tissues¹²⁾.

To confirm the tumor suppressive effect and the mechanisms of p16^INK4A gene in non-small cell lung cancer, we transfected p16^INK4A gene into cell lines of this cancer via lipofectin. p16(−) NCI-H441 cell line transfected with pRC-CMV-p16 showed the formation of p16:cdk4 complex and decreased phosphorylated Rb protein, while the control cell line did not. These results are consistent with those of Sandig et al., who studied the expression of pRB in a hepatocellular carcinoma cell line¹³⁾. pRB has been shown to play a protective role in cell apoptosis¹⁴⁾. When its levels are sufficiently reduced, this role can no longer be maintained, and this may explain part of the tumor suppressor function of p16. Clonogenic assay demonstrated that the extent of colony formation was significantly less in p16(−) NCI-H441 and NCI-H157 cell lines transfected with pRC-CMV-p16 than in the control cell line. This study shows that the expression of p16 protein in gene-transfected NSCLC cell lines, in which p16 is absent, leads to p16:cdk4 complex formation and a subsequent decrease in phosphorylated pRb protein levels, and leads ultimately to tumor suppressive effects.

Using pRC/CMV plasmid vector, Okamoto et al. showed that when p16^INK4A gene transfer was involved, colony formation by esophageal cancer cell lines decreased¹²⁾. Arap et al. demonstrated the growth suppressive effect of p16^INK4A gene in p16(−) glioma cell lines¹⁵⁾, and this finding is in agreement with those of other reports.^{16, 17)} Though this effect was not observed in glioma cell lines which express wild-type p16 protein¹⁵⁾. It has been suggested that CDK4 or cyclinD may be amplified or overexpressed^{18, 19)}, or a G1/S cell cycle pathway such as p53-p21^WAF1 may be inactivated in cases of glioma with wild-type p16 expression¹⁵⁾. This suggests that, for the general use of gene therapy for the treatment of cancer, combination gene therapy involving p53 and p16, for example, may be necessary.

Tumor suppressor gene therapy requires efficient delivery of therapeutic genes and a high level of expression of the newly transduced gene in the tumor. The adenovirus is an efficient expression vector that can be produced in high titers²⁰⁾. Its expression time in the host cell is short, and it is thus suitable for use in gene replacement strategies involved in cancer treatment. For the clinical trial of p16 in cancer gene therapy, adenovirus vector is one of the suitable candidates. A few in vitro and animal studies on p16 in the adenovirus vector have been published recently^{16, 17, 21)}.

It was difficult to obtain cell lines in which the expression of p16 protein after transfection of p16^INK4A gene in NSCLC cell lines was stable. Because p16-transfected cells which overexpress p16 do not usually survive, we could not obtain data relating to flow cytometric cell cycle analysis or quantitative cytotoxicity tests. This difficuty is a common problem in the study of tumor suppressor genes and, to overcome it, an inducible promotor is usually used in the cloning of expression vectors.

In this study, we confirmed in vitro that after transfection of p16^INK4A gene to a cell line which has wild-type pRB but does not express p16, p16 formed a complex with cdk4, phosphorylation of pRB decreased, and there was decreased colony formation. This suggests that the p16^INK4A gene can be a candidate for gene therapy in cases of NSCLC in which p16^INK4A gene is inactivated.