The p53 protein directly regulates the expression of the WAF1 (wild-type p53-activated fragment 1) protein which is a cyclin-dependent kinase inhibitor (CDK1). DNA damaging agents such as ionizing or UV radiation, and some chemical agents induce WAF1 in wild-type p53 containing cells, thereby halting cell cycle progression. WAF1 expression is also induced through a p53-independent pathway. Tumor necrosis factor alpha (TNF alpha) is a cytotoxic/cytostatic compound for some human cancer cells. We examined a series of myeloid leukemic cell lines that expressed either no p53 (HL-60, K562) or mutant inactive p53 (KG-1, KCL22,THP-1, U937). The KG-1, HL-60, K562, and KCL22 myeloid leukemic cells increased their levels of WAF1 mRNA in the presence of TNF alpha. We focused on KG-1 cells to determine how TNF alpha modulated WAF1 expression. WAF1 mRNA increased in a dose-dependent manner in the cells after exposure to increasing concentrations of TNF alpha, and this increase occurred in the absence of new protein synthesis. An increase of WAF1 protein and a concominant decrease of cyclin-dependent kinase 2 activity also was found in KG-1 cells. Flow cytometry using 5-bromo-2'-deoxyuridine showed an increase in the proportion of TNF alpha- treated KG-1 cells in the G0/G1 phase of the cell cycle. TNF alpha enhanced the rate of WAF1 transcription only 1.4 fold in TNF alpha-treated KG-1 cells as compared to untreated cells. Notably, however, the half-life (t 1/2) of WAF1 mRNA in TNF alpha-treated cells was 2.5 hours as compared to 0.5 hours in untreated cells. These results indicate that TNF alpha increases WAF1 levels at least in part via a postttranscriptional stabilization of the mRNA; and TNF alpha may mediate its cytostatic effects through WAF1 in some cell types.

The protein p27KIP1 belongs to a recently identified family of proteins termed cyclin-dependent kinase inhibitors (CDKIs). These proteins play an important role in regulating cell-cycle progression and loss of their function has been implicated in tumorigenesis. Transforming growth factor beta (TGF-beta) may induce cell growth arrest through p27 activation. TGF-beta often loses its ability to arrest growth of transformed cells; this could potentially occur through a defect in p27. To determine the role of p27 in tumorigenesis, we examined its mutational status in 74 non-Hodgkin's lymphomas (NHLs) (52 of B-cell phenotype, 22 of T-cell phenotype), 5 lymphoma cell lines, and 42 adult T-cell leukemias/lymphomas (ATLs) using polymerase chain reaction- single strand conformational polymorphism (PCR-SSCP) and Southern blot analyses. A nonsense mutation (stop codon) that could result in expression of a truncated nonfunctional p27 protein was detected at codon 76 in one case of acute lymphomatous ATL, but not in matched normal tissues. Previously undescribed polymorphisms were also identified at codon 109 in the lymphomas and at codon 55 in the ATLs. Two homozygous deletions of the p27 gene were detected in one case of B- immunoblastic NHL and in one case of acute ATL by Southern blot hybridization. These results indicate that p27 gene alterations are rare events in NHLs and ATLs, but may play an important role in the pathogenesis of some hematologic malignancies.

The tumor suppressor genes p16INK4A and p15INK4B map to the 9p21 chromosomal locus and are either homozygously deleted or mutated in a wide range of human cancer cell lines and tumors. Although chromosome 9 abnormalities have been described in non-Hodgkin's lymphomas (NHLs), to date, the mutational status of these genes has not been determined for these malignancies. A total of five cell lines and 75 NHLs were examined for homozygous deletions or point mutations in the coding regions of both the p15 and p16 genes using Southern blot and/or polymerase chain reaction-single-strand conformation polymorphism analyses. Homozygous deletions of either the p16 gene or both the p15 and p16 genes were observed in one diffuse large B-cell lymphoma cell line and two uncultured lymphomas consisting of one large B-cell and one mixed T-cell lymphoma. In contrast, point mutations were not detected in either the cell lines or lymphomas. These results indicate that the rate of alterations in the p15 and p16 genes is low for lymphomas, but loss of p16 and/or p15 may be involved in the development of some lymphomas.

CDK6 activity.We tested the efficacy of our system by expressing the wt orEvidence from studies on both cell lines and human tumors deletion of the p16 gene, frequently point mutations are