Kari Punnonen

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. Int. J. Cancer 74:255‐259, 1997.

Antioxidant enzyme activities and oxidative stress in human breast cancer

We have analysed products of lipid peroxidation reactions and activities of antioxidant enzymes in cancerous breast tissue and in corresponding reference tissue. In addition, the serum lipid peroxidation and peroxyl-radical-trapping capacity of breast cancer patients were compared to those of healthy subjects. A total of 23 patients with breast cancer participated in this study. In the cancerous tissue, catalase activity was lower than in the reference tissue, while the activities of superoxide dismutase, glutathione peroxidase and the hexose monophosphate shunt were elevated. The content of thiobarbituric-acid-reactive material was slightly lower in the cancerous tissues, but the levels in serum were found to be elevated in patients with breast cancer. The amounts of conjugated diene double bonds were essentially equal both in the cancerous and in the reference tissue. Moreover, in breast cancer patients the serum levels of diene conjugation and the peroxyl-radical-scavenging capacity did not differ from those measured in healthy subjects. This study indicates that the antioxidant defence system is altered in cancerous breast tissues, but does not support the hypothesis suggesting that formation of lipid peroxides in the tumour tissue itself is of primary importance in the carcinogenesis.

In-vivo effects of solar-simulated ultraviolet irradiation on antioxidant enzymes and lipid peroxidation in human epidermis

The effects of solar‐simulated UV‐irradiation on the activity of antioxidant enzymes and the amount of diene conjugation were studied in human epidermis in vivo. A single dose of UV‐irradiation was found to result in a transient reduction in superoxide dismutase activity and this was followed by increased amounts of conjugated diene double bonds, an index for oxidative stress. This suggests that in‐vivo exposure of human epidermis to solar‐simulated UV‐irradiation causes changes in the enzymic antioxidant defence system which, in turn, are accompanied by increased level of oxidative stress.

Regulation of copper/zinc and manganese superoxide dismutase by UVB irradiation, oxidative stress and cytokines

We have examined the effects of UVB irradiation, oxidative stress and cytokines on the antioxidant enzymes copper/zinc and manganese superoxide dismutase (CuZnSOD and MnSOD) in HeLa cells. A single dose of UVB irradiation regulated dose-dependently the expression of the 4 kb transcript of MnSOD although it did not have any significant effect on MnSOD enzymatic activity. In contrast, UVB irradiation reduced both the enzymatic activity and the expression of the 0.7 and 0.9 kb mRNA transcripts of CuZnSOD. The cytokines TNF-alpha (1 ng ml-1 and 10 ng ml-1) and IL-6 (100 U ml-1) induced MnSOD activity, and TNF-alpha also upregulated MnSOD mRNA expression. Interestingly, genistein, a soy isoflavone and a tyrosine kinase inhibitor, was able to inhibit the induction of Mn-SOD activity and mRNA expression by TNF-alpha. Enzymatic CuZnSOD activity was depressed by a high dose of H2O2 while IL-6 or TNF-alpha had no effect on CuZnSOD activity. Our results demonstrate that, in addition to enzyme activity level, UVB irradiation can regulate the superoxide dismutases at the mRNA level. We also suggest that UVB irradiation, oxidative stress and cytokines regulate differentially CuZnSOD and MnSOD, and that the activities and expression of these antioxidant enzymes are controlled by distinct mechanisms.

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

Abstract 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/cm 2 ). The expression of bcl -2 mRNA was found to decrease after a single dose of UVB radiation (doses 10–200 mJ/ cm 2 ). 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/cm 2 ), 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.

Differential Regulation of the AP-1 Family Members by UV Irradiation In Vitro and In Vivo

We have examined the effect of UVB and solar-simulated irradiation on the expression of the AP-1 family of transcription factors and the cytokine IL-6 both in cell cultures and in human skin in vivo. UVB irradiation potently induced c-jun, junB and c-fos mRNA levels in vitro in HaCaT cells. IL-6 mRNA was induced in response to UVB irradiation 2-3 h later than c-jun, junB and c-fos mRNAs. In human skin in vivo, solar-simulated irradiation induced transiently junB expression. Genistein, a tyrosine kinase inhibitor, augmented the induction of c-jun and junB by UVB irradiation in HaCaT cells. The results of this study provide evidence that in addition to c-jun and c-fos, junB is also an essential component of the human UV-response. This study also suggests that UVB irradiation regulates the AP-1 family by several mechanisms and that the signalling mechanisms of UVB irradiation are considerably different from the ones used by UVC irradiation.

Activities of antioxidant enzymes and lipid peroxidation in endometrial cancer

Antioxidant enzyme activities and lipid peroxidation were analysed in normal endometrium and endometrial cancer tissues from Finnish and Japanese patients. The catalase and glutathione peroxidase activities of normal endometrium were significantly lower in Finns than in Japanese. Lipid peroxidation was slightly higher in endometrial cancer as compared with normal endometrium both in the Finns and in the Japanese. When cancer tissues were compared with normal endometrium both in Finns and Japanese the activity of superoxide dismutase was significantly lower in cancer tissue than in normal endometrium. In Finns glutathione S-transferase activity was also lower in endometrial cancer tissue than in normal endometrium, and a similar tendency was also found in Japanese. This study suggests that endometrial cancer tissue is associated with an impaired enzymic antioxidant defence system.

Chronic UVB irradiation induces superoxide dismutase activity in human epidermis in vivo

In order to study the effects of repeated UVB exposures on the epidermal antioxidant defence system, we obtained epidermis samples from male volunteers who were exposed to chronic UVB irradiation. Chronic UVB irradiation was shown to be accompanied by induction of epidermal superoxide dismutase (SOD) activity in vivo, while the activities of the other antioxidant enzymes were not significantly changed. The repeated exposure of the epidermis to UVB irradiation was not accompanied by accumulation of products of lipid peroxidation reactions. As superoxide dismutase is of major importance in scavenging the reactive oxygen species, the UVB-induced changes in SOD activity might provide the epidermis a way of defending itself against the effects of chronic UVB irradiation.

Phospholipids and fatty acids in breast cancer tissue.

SummaryThe fatty acid composition of fractionated phospholipids and neutral lipids was analyzed in human breast cancer tissues and the surrounding, apparently healthy tissue. In the cancer tissues the relative amounts of unsaturated fatty acids were increased in all the phospholipid subclasses analyzed. The differences were more marked in phosphatidylethanolamine than in the other phospholipid fractions and, furthermore, the relative amount of phosphatidylethanolamine was increased in cancerous tissue. In blood-erythrocyte phospholipids, no differences in fatty acid composition could be found between breast cancer and control patients. The present study suggests that the lipid composition of cancerous breast tissues differs from that of the surrounding tissue and may be involved in carcinogenesis.

Incorporation and distribution of dihomo-γ-linolenic acid, arachidonic acid, and eicosapentaenoic acid in cultured human keratinocytes

Human keratinocytes in culture were labelled with 14C-dihomo-gamma-linolenic acid, 14C-arachidonic acid or 14C-eicosapentaenoic acid. All three eicosanoid precursor fatty acids were effectively incorporated into the cells. In phospholipids most of the radioactivity was recovered, in neutral lipids a substantial amount, and as free unesterified fatty acids only a minor amount. The most of the radioactivity was found in phosphatidylethanolamine which was also the major phospholipid as measured by phosphorous assay. The incorporation of dihomo-gamma-linolenic acid and arachidonic acid into lipid subfractions was essentially similar. Eicosapentaenoic acid was, however, much less effectively incorporated into phosphatidylinositol + phosphatidylserine and, correspondingly, more effectively into triacylglycerols as compared to the two other precursor fatty acids. Once incorporated, the distribution of all three precursor fatty acids was relatively stable, and only minor amounts of fatty acids were released into the culture medium during short term culture (two days). Our study demonstrates that eicosanoid precursor fatty acids are avidly taken up by human keratinocytes and esterified into membrane lipids. The clinical implication of this finding is that dietary manipulations might be employed to cause changes in the fatty acid composition of keratinocytes.