Cell cycle regulators p27 and pRb in lymphomas - correlation with histology and proliferative activity.

The cell cycle is a complex event in which multiple regulator-proteins participate. The G(1)/S checkpoint of the cell cycle is controlled by pRb protein, which functions in its hypophosphorylated form as a negative regulator of growth. p27 (Kip1), a member of CIP/KIP family of cyclin inhibitory proteins, participates in inhibition of forming complexes that allow pRb to phosphorylate and lead the cell into mitosis. The expression of these important cell cycle regulator proteins was studied in a total of 96 non-Hodgkin's lymphoma (NHL) samples, which were classified according to the REAL classification. The expression of p27, pRb and the cell proliferation marker Ki-67 (MIB-1) was evaluated in lymphomas using immunohistochemistry. This study showed that there were coordinate changes in the expression of p27 and pRb in NHL. When compared to low-grade lymphomas, high-grade lymphomas showed significantly reduced expression of p27 and inversely pRb expression was increased (P< 0.001). Increase in expression of Ki-67 was parallel with pRb expression, and was mainly seen in cells that lacked p27 expression (P< 0.0001). This study suggests that changes in the control of the cell cycle closely relate to the pathobiology of NHL.

Analysis of G(1)/S checkpoint regulators in metastatic melanoma.

We have analyzed the expression of the CDKN1A (p21(CIP1)), CDKN1B (p27(Kip1)), TP53, RB1 and MDM2 proteins and tumor cell proliferation by immunohistochemical staining in 59 cases of metastatic melanoma. The genomic status of the CDKN2A (INK4-ARF, p16/p14(ARF)), CDKN2B (p15) and CDKN2C (p18) genes was determined by PCR-SSCP (single-strand conformation polymorphism) in 46 of these cases. These results were correlated with various clinico-pathological parameters, including the outcome of combined chemoimmunotherapy. We found positive correlations between the expression of CDKN1A and MDM2 (r = 0.5063, P = 0.001), between the expression of CDKN1B and RB1 (r = 0.5026, P = 0.001), and between RB1 expression and tumor cell proliferation (0.5564, P<0.001). Two mutations in the CDKN2A (p16) gene were detected, including a novel base change AAC-->ATC (Asn to Ile) at codon 71, that also changes the codon 85 of the alternative reading frame gene p14(ARF) from CAA to CAT (Gln to His). Homozygous deletion at exon 2 of the CDKN2A (INK4-ARF) gene was detected in six cases. In seven cases, the 540C-->G polymorphism in the 3'UTR of the CDKN2A (p16) gene was found in linkage disequilibrium with the 74C-->A polymorphism in intron 1 of the CDKN2B gene (P < 0.0001). These cases had significantly lower expression of the TP53 protein (P = 0.0032). Both 540C-->G and 580C-->T polymorphisms in the 3'UTR of the CDKN2A (p16) gene were associated with significantly shorter progression time from primary to metastatic disease (P = 0.0071). We conclude, that although none of the analyzed cell cycle regulators could be singled out as a major prognostic factor, G(1)/S checkpoint abnormalities remain one of the most significant factors in the development of malignant melanoma.

UV irradiation induces downregulation of bcl-2 expression in vitro and in vivo.

Recently, the proto-oncogenes bcl-2 and bax have emerged as important regulators of the apoptotic form of cell death. We examined UV irradiation-elicited apoptosis and regulation of bcl-2 and bax expression both in vivo in human skin and in vitro in HeLa cells. Using flow cytometric analysis, HeLa cells were found to undergo apoptosis at the 12-h time-point after exposure to UVB irradiation (100 mJ/cm2). The expression of bcl-2 mRNA was found to decrease after a single dose of UVB radiation (doses 10-200 mJ/cm2). In contrast, the expression of bax mRNA was not significantly changed. When human skin was irradiated with a single dose of solar-simulated radiation (40 mJ/cm2), Bcl-2-positive cells were significantly reduced in the epidermis at the 3- and 6-h time-points. Our results suggest that UV irradiation downregulates bcl-2 expression both in vitro at the mRNA level and in vivo at the protein level, and that downregulation of bcl-2 constitutes a mechanism of potential importance in UV-induced apoptosis in human epidermis.

Selective deletion of exon 1 beta of the p19ARF gene in metastatic melanoma cell lines.

The INK4A locus on 9p21 is deleted or rearranged in a large number of human cancers. The locus encodes two unrelated and independently acting negative cell-cycle regulators, p16 and p19ARF, arising in alternate reading frames from a partly shared sequence. We analyzed five human melanoma cell lines for deletions at the INK4 loci and flanking microsatellite markers on 9p21. All the cell lines displayed deletions of varying sizes. The metastatic cell line IGR-1 showed a large deletion between the markers D9S736 and D9S171. In the cell lines WM-115 and WM-266-4, the deletion included exon 1alpha of p16, exon 1beta of p19ARF, and exon 2 of the INK4B (p15) gene. Two cell lines, SK-MEL-5 and A2058, had deletions confined to exon 1beta and the microsatellite marker D9S942. RT-PCR experiments showed the presence of the p16 and p15 transcripts and absence of p19ARF expression in both SK-MEL-5 and A2058 cell lines. The selective loss of the exon 1beta of p19ARF and retention of the p16 and p15 genes and their expressions in these two cell lines support the putative tumor suppressor role for the alternate reading frame p19ARF gene.

Loss of expression of the p16INK4/CDKN2 gene in cutaneous malignant melanoma correlates with tumor cell proliferation and invasive stage.

The G1/S checkpoint of the cell cycle is regulated by p16, p53 and RB tumor suppressor genes. Loss of expression of the p16INK4 tumor suppressor protein, the product of the CDKN2 gene, has been associated with a wide variety of human malignancies. Mutations, loss of heterozygosity and deletions of the CDKN2 locus have been reported in sporadic and familial cutaneous malignant melanomas (CMM). To investigate the role of the alterations of p16 expression in melanoma, we evaluated by immunohistochemistry the p16 expression and cell proliferation in 79 primary CMM and 10 benign melanocytic nevi (BMN). Forty-six melanomas (58%) and all BMN were found to be p16 positive; 33 melanomas (42%) were considered p16 negative. The extent of invasion according to Clark was significantly higher in p16-negative tumors than in p16-positive tumors. Cell proliferation as expressed by the proportion of positive cells in Ki-67 immunostaining was found to be significantly higher in p16-negative tumors than in p16-positive tumors, although there was no significant difference in the mitotic index between p16-positive and p16-negative tumors. In p16-positive tumors, the number of Ki-67-positive cells correlated with the mitotic index; in p16-negative tumors, there was no correlation between these parameters. Our data suggest that loss of p16 expression is more common in advanced melanomas, and that G1/S checkpoint regulation is disrupted in p16-negative melanomas. Our results show that loss of p16 expression is a common event in primary melanomas, which further substantiates the role of p16 as a major tumor suppressor.