Loss of expression of protein kinase C beta is a common phenomenon in human malignant melanoma: a result of transformation or differentiation?

As with most cancers, the aetiology of human cutaneous melanoma is likely to be multifactorial and to include the accumulation of irreversible alterations in an unknown number of genes. Elucidating this molecular progression necessitates both the identification of genetic perturbations at each clinically relevant stage, and the assessment of their impact on the normal melanocyte. The observation that the epidermal melanocyte, in contrast to metastatic melanoma cells, requires activation of the protein kinase C (PKC) pathway to facilitate growth in vitro indicates that one or more isoforms (or substrates) of this large and complex family of proteins are among those that undergo alteration during the development of malignant melanoma. Consequently, a number of studies have investigated the expression of various PKC family members in both melanocyte and melanoma cell lines, without a consensus of opinion as to which isoforms are of biological significance in melanoma development and progression. The present study involved a comprehensive evaluation of the PKC profile in normal melanocytes and in 16 metastatic melanoma cell lines. The results show that the major difference in isoform expression between epidermal melanocytes and melanoma cells is the loss of PKCbeta protein expression in 90% of melanoma cell lines. Examination of PKCbeta in benign and malignant melanocytic lesions revealed that this protein is either downregulated or absent in both naevi and metastatic melanomas. We conjecture that, although the loss of PKCbeta expression is a common phenomenon in malignant melanocytes, it may be related more to a normal process of melanocytic differentiation than to malignant transformation.

Protein kinase C and its isoforms in human breast cancer cells: relationship to the invasive phenotype.

Total protein kinase C (PKC) activity and the expression of 9 isoforms were determined in the estrogen receptor (ER) positive MCF-7 human breast cancer cell line, this line transfected to overexpress either PKC-á or erbB2, and in 3 ER negative breast cancer cell lines. Relationships were sought between PKC and the expression of E-cadherin, alpha-catenin, and vimentin, and urokinase-type plasminogen activator (uPA). In general, PKC enzymic activity and the conventional isoforms PKC-alpha and -gamma were higher in the ER negative, compared with the ER positive, cell lines. Over-expression of PKC-alpha by MCF-7 cells, with ER loss, was associated with the emergence of PKC- expression and a relatively high level of PKC-gamma, features typical of cells with increased proliferation rates; there was also a loss of PKC-delta, consistent with acquisition of the metastatic phenotype. Transfection to overexpress erbB2, with ER retention and slowed growth, produced a decrease in PKC-alpha and -gamma. Vimentin was expressed by the ER negative MDA-MB-231, MDA-MB-435 and PKC-alpha-transfected MCF-7 cells; they also showed loss of E-cadherin and, apart from MDA-MB-435 cells, high levels of uPA secretion. The ER negative SKBr-3 cell line was exceptional in that it had relatively low total PKC activity, low PKC-alpha and -gamma expression and no emergence of vimentin despite loss of E-cadherin expression. Compared with the other two ER negative cell lines, both the SKBr-3 and MDA-MB-435 cells had low PKC activity and uPA secretion. These results are consistent with the involvement of PKC, and notably the conventional isoforms, in the development of the metastatic phenotype, and specifically with the loss of E-cadherin and acquisition of vimentin expression, and the enhanced production of uPA.

Topoisomerase II expression and VM-26 induction of DNA breaks during spermatogenesis in Xenopus laevis.

The relative content of topoisomerase II (topo II) and the induction of topo-II-mediated DNA damage and cellular abnormalities have been characterized in developing spermatogenic cells of Xenopus laevis to gain an insight into the role of topo II during spermatogenesis. Decatenation assays identified topo II activity in nuclear extracts from spermatocytes and pre-elongate spermatids, but not in extracts from elongate spermatids or sperm. Extracts from early-mid spermatids contained 14% (per cell) of the decatenation activity found in spermatocyte extracts. Immunoblots of SDS extracts from whole cells and nuclei from both spermatocytes and pre-elongate spermatids, but not elongate spermatids or sperm, resolved a 180 kDa polypeptide that reacts with polyclonal antisera to Xenopus oocyte topo II, an antipeptide antibody (FHD29) to human topo II alpha and beta, and an antipeptide antibody to human topo II alpha, suggesting homology between Xenopus spermatogenic cell topo II and mammalian topo II alpha. Immunofluorescence microscopy of topo II in testis cryosections revealed the presence of topo II in nuclei of all spermatogenic stages, but not in sperm. The relative levels of topo II estimated from fluorescence intensity were highest in spermatogonia and spermatocytes, then early-mid spermatids, followed by elongate spermatids and somatic cells. Incubation of isolated spermatogenic cells with teniposide (VM-26), a topo II-targetted drug, resulted in a dose-dependent induction of DNA breaks in all spermatocytes and spermatid stages to nuclear elongation stages, as analyzed by alkaline single cell gel electrophoresis. Addition of 0.5-50 microM VM-26 to spermatogenic cell cultures for 27 hours resulted in stage-dependent abnormalities. Mid-late spermatid stages were relatively resistant to VM-26-induced damage. In contrast, meiotic division stages were arrested and spermatogonia B were killed by VM-26, and VM-26 induced abnormal chromosome condensation in pachytene spermatocytes. The results of these studies show that cellular levels of topo II are stage-dependent during spermatogenesis, that most spermatogenic stages are sensitive to topo II-mediated DNA damage, and that spermatogonia B, meiotic divisions and pachytene spermatocytes are particularly sensitive to induction of morphological abnormalities and cell death during acute exposure to topo II-targetted drugs.

Comparative aspects of spermatogenic cell metabolism and Sertoli cell function in Xenopus laevis and mammals.

Due to the relative dearth of data regarding somatic cell-germ cell interactions in the testes of non-mammalian chordates, functional homologies between Sertoli cells from diverse organisms have been difficult to assess. However, recent developments in non-mammalian testis cell and organ culture techniques have provided experimental approaches to compare Sertoli cell-germ cell interactions in different vertebrates. Data from in vitro analyses of Sertoli cell-germ cell interactions are presented to suggest that Sertoli cells from rodents and the frog Xenopus laevis have similarities in supporting energy metabolism and glutathione metabolism in spermatogenic cells. Comparative in vitro analyses of Sertoli cell functions should provide further insights into the evolution of cell-cell interactions in the testes.

The role of glutathione in the aerobic radioresponse. I. Sensitization and recovery in the absence of intracellular glutathione.

The effect of changes in both the intracellular glutathione (GSH) concentration and the concentration of extracellular reducing equivalents on the aerobic radiosensitization was studied in three cell lines: CHO-10B4, V79, and A549. Intracellular GSH was metabolically depleted after the inhibition of GSH synthesis by buthionine sulfoximine (BSO), while the extracellular environment was controlled through the replacement of growth medium with a thiol-free salt solution and in some experiments by the exogenous addition of either GSH or GSSG. Each of the cell lines examined exhibited an enhanced aerobic radioresponse when the intracellular GSH was extensively depleted (GSH less than 1 nmol GSH/10(6) cells after 1.0 mM BSO/24 h treatment) and the complexity of the extracellular milieu decreased. Although the addition of oxidized glutathione (5 mM GSSG/30 min) to cells prior to irradiation was without effect, much or all of the induced radiosensitivity was overcome by the addition of reduced glutathione (5 mM GSH/15 min). However, the observation that the exogenous GSH addition restores the control radioresponse without increasing the intracellular GSH concentration was entirely unexpected. These results suggest that a number of factors exert an influence on the extent of GSH depletion and determine the extent of aerobic radiosensitization. Furthermore, the interaction of exogenous GSH with--but without penetrating--the cell membrane is sufficient to result in radiorecovery.

Glutathione depletion, radiosensitization, and misonidazole potentiation in hypoxic Chinese hamster ovary cells by buthionine sulfoximine.

Buthionine sulfoximine (BSO) inhibits the synthesis of glutathione (GSH), the major nonprotein sulfhydryl (NPSH) present in most mammalian cells. BSO concentrations from 1 microM to 0.1 mM reduced intracellular GSH at different rates, while BSO greater than or equal to 0.1 mM (i.e., 0.1 to 2.0 mM), resulting in inhibitor-enzyme saturation, depleted GSH to less than 10% of control within 10 hr at about equal rates. BSO exposures used in these experiments were not cytotoxic with the one exception that 2.0 mM BSO/24 hr reduced cell viability to approximately 50%. However, alterations in either the cell doubling time(s) or the cell age density distribution(s) were not observed with the BSO exposures used to determine its radiosensitizing effect. BSO significantly radiosensitized (ER = 1.41 with 0.1 mM BSO/24 hr) hypoxic, but not aerobic, CHO cells when the GSH and NPSH concentrations were reduced to less than 10 and 20% of control, respectively, and maximum radiosensitivity was even achieved with microM concentrations of BSO (ER = 1.38 with 10 microM BSO/24 hr). Furthermore, BSO exposure (0.1 mM BSO/24 hr) also enhanced the radiosensitizing effect of various concentrations of misonidazole on hypoxic CHO cells.