Sikha Rauth

Effect of double-strand breaks on homologous recombination in mammalian cells and extracts.

We examined the effect of double-strand breaks on homologous recombination between two plasmids in human cells and in nuclear extracts prepared from human and rodent cells. Two pSV2neo plasmids containing nonreverting, nonoverlapping deletions were cotransfected into cells or incubated with cell extracts. Generation of intact neo genes was monitored by the ability of the DNA to confer G418r to cells or Neor to bacteria. We show that double-strand breaks at the sites of the deletions enhanced recombination frequency, whereas breaks outside the neo gene had no effect. Examination of the plasmids obtained from experiments involving the cell extracts revealed that gene conversion events play an important role in the generation of plasmids containing intact neo genes. Studies with plasmids carrying multiple polymorphic genetic markers revealed that markers located within 1,000 base pairs could be readily coconverted. The frequency of coconversion decreased with increasing distance between the markers. The plasmids we constructed along with the in vitro system should permit a detailed analysis of homologous recombinational events mediated by mammalian enzymes.

Melanoma cells can tolerate high levels of transcriptionally active endogenous p53 but are sensitive to retrovirus-transduced p53

Malignant melanomas are frequently characterized by elevated levels of wild-type p53, suggesting that p53 function could be suppressed by a mechanism different from p53 mutation. We analysed the functionality of the p53-signaling pathway in a panel of seven human melanoma cell lines consisting of one p53-deficient line, two lines with mutant p53, and four lines expressing wild-type p53. Only lines with wild-type p53 were characterized by elevated levels of endogenous p21, high activity of p53-responsive reporters and accumulation of p53 in response to genotoxic stress, common properties of functional p53. The presence of wild-type p53 was associated with depletion or loss of p14ARF and p16 expression. The levels of p33ING1b and p24ING1c, two major products of Ing1 locus and putative coregulators of p53, were elevated in all cell lines tested; however, ectopic expression of either ING1 isoform had no effect on cell proliferation. All lines retained expression of Apaf-1, and all but one remained sensitive to ectopic expression of retrovirus-transduced p53. Our data indicate that regardless of abnormally high levels of p53 in melanomas, their p53 remains competent in transactivation of its targets, and, if highly overexpressed, capable of growth inhibition. Hence, the p53 pathway in malignant melanomas can be considered for pharmacological targeting and anticancer gene therapy.

Suppression of tyrosinase gene expression by bromodeoxyuridine in Syrian hamster melanoma cells is not due to its incorporation into upstream or coding sequences of the tyrosinase gene

Abstract5-Bromodeoxyuridine (BrdU), a thymidine analog, suppresses melanogenesis in Syrian hamster melanoma cells. Tyrosinase, which is the key enzyme for the synthesis of melanin, is suppressed by exposure to BrdU, and the drop in enzyme activity is correlated with a drop in tyrosinase mRNA level. In order to investigate whether suppression of tyrosinase mRNA by BrdU is due to BrdU substitution into coding sequences or upstream sequences of the tyrosinase gene, we carried out stable and transient transfection assays with constructs containing either the human tyrosinase cDNA sequence under the control of a nontyrosinase promoter or a chloramphenicol acetyltransferase (CAT) reporter gene under the control of 5′ flanking sequences of the mouse tyrosinase gene. When the plasmid containing the tyrosinase cDNA was stably transfected into mouse fibroblasts, tyrosinase activity in the transfectants was not suppressed by BrdU. Since BrdU would be incorporated into the tyrosinase cDNA integrated in these transfectants, the results suggest that BrdU suppression of tyrosinase gene expression is not due to its incorporation into coding sequences of the tyrosinase gene. When plasmids with tyrosinase regulatory sequences were transfected into melanoma cells for transient expression assays, CAT gene expression was suppressed by BrdU. Because the CAT plasmids do not contain a mammalian origin of replication and should not replicate under the conditions of transient transfection, BrdU would not be incorporated into the DNA of those plasmids. Therefore, these results suggest that the suppression of tyrosinase gene expression by BrdU also is not due to the incorporation of BrdU into upstream sequences of the tyrosinase gene.

Bromodeoxyuridine- and cyclic AMP-mediated regulation of tyrosinase in Syrian hamster melanoma cells

The thymidine analog 5-bromodeoxyuridine (BrdU) suppresses pigmentation and tyrosinase activity in Syrian hamster melanoma cells W1-1-1. Studies on the molecular mechanism of suppression of pigmentation indicated that BrdU treatment affects the level of tyrosinase gene transcripts. No detectable tyrosinase message was found by Northern blot analysis in cells cultured in the presence of BrdU at concentrations even as low as 0.2 µM. The level of tyrosinase mRNA was found to reflect the level of pigmentation and tyrosinase activity. Studies with dibutyryl cyclic AMP (cAMP) showed that it inhibited pigment synthesis in W1-1-1 cells. With increasing concentrations of cAMP ranging from 10 µM to 300 µM, pigmentation and tyrosinase activity decreased progressively. This inhibition was found to be associated with a corresponding decrease in the level of tyrosinase mRNA. W1-1-1 cells were found not to respond to melanocyte stimulating hormone (MSH). There was no change in pigmentation, tyrosinase activity, or tyrosinase mRNA level in W1-1-1 cells in the presence of MSH. Similarly, theophylline, a phosphodiesterase inhibitor, had no effect on pigmentation or tyrosinase activity in W1-1-1 cells.

GC-rich murine adenosine deaminase gene promoter supports diverse tissue-specific gene expression

The murine adenosine deaminase (ADA) gene has a GC-rich promoter that is structurally typical of many mammalian “housekeeping” gene promoters. The ability of theADA gene promoter to support diverse tissue-specific gene expression was investigated EndogenousADA gene expression in different mouse tissues was found to vary over a >3000-fold range in a highly complex pattern. This range of expression was also observed in cultured human cell lines derived from different tissues. The ADA levels in all tissues and cell lines examined correlated closely with steady-state ADA mRNA levels. Several of the mouse tissues examined also showed stage-specific variation during postnatal development. In order to determine whether tissue-specificADA expression was controlled by cis-acting sequences upstream of the coding region, constructs containing a reporter gene regulated by the ADA genes 5′ flanking sequences were used to generate transgenic mice. All transgene-expressing mice obtained showed diverse reporter gene expression in the tissues analyzed. Our results demonstrate that both in vivo and in the context of an integrated transgene this GC-rich promoter can support highly diverse gene expression in all tissues of the animal.

Suppression of tumorigenic and metastatic potentials of human melanoma cell lines by mutated (143 Val-Ala) p53

Metastatic melanoma, compared with other cancers, appears to be unusual because of its low frequency of p53 mutations and prevalence of wild-type p53 protein in advanced malignancy. Here, we examined the effects of wild-type and mutated p53 (143 Val-Ala) on tumorigenic and metastatic potential of two human melanoma cell lines. The cell line UISO-MEL-4 contains wild-type p53 and is tumorigenic, whereas UISO-MEL-6 lacks p53 and produces lung and liver metastasis upon s.c. injection into athymic mice. Our study showed that UISO-MEL-4 stably transfected with wild-type p53 cDNA driven by cytomegalovirus promoter-enhancer sequences expressed high levels of p53 and p21 and formed s.c. tumours in vivo. Mutated p53 (143 Val-Ala) expression, on the other hand, inhibited tumour growth in 50% of cases and produced significantly slower growing non-metastatic tumours. Reduced tumour growth involved necrotic as well as apoptotic cell death. Inhibition of tumour growth was abrogated by the addition of Matrigel (15 mg ml(-1)). With UISO-MEL-6 cells, stably transfected with mutant p53, tumour growth was delayed and metastasis was inhibited. In soft agar colony formation assay, both wild-type and mutant p53 transfectants reduced anchorage-independent colony formation in vitro. These data suggest that mutated (143 Val-Ala) p53, which retains DNA binding and some of the transactivation functions of the wild-type p53 protein, suppresses tumorigenic and metastatic potentials of human melanoma cell lines in vivo.

Expression of DNA transferred into mammalian cells

There are several methods to introduce purified DNA into mammalian cells. These include microinjection into the nuclei of the recipient cells and complexing the DNA with facilitating agents such as calcium-phosphate. After it enters the nucleus, the DNA is expressed in a large proportion of the cells. This expression is dependent upon cis-acting sequences that are recognized by the mammalian transcriptional and translational machinery. In a smaller proportion of cells, the exogenous DNA becomes covalently integrated into the host cell DNA at random sites. Non-selectable genes can be introduced into mammalian cells by ligating them to a selectable marker or mixing the DNA with carrier DNA containing a selectable marker. The DNA that is introduced into mammalian cells can be rescued for examination and analysis. These gene transfer methods have already proven to be useful in identification of sequences which are necessary for normal gene expression as well as gene regulation. In addition a number of genes have been isolated using gene transfer methods. DNA mediated gene transfer holds much promise to target genes to specific sites in the chromosomes, to understand mechanisms of mammalian development and cell differentiation and is expected to provide a method to produce important and novel gene products that may be used for diagnostic and therapeutic purposes.

Chromosome abnormalities in metastatic melanoma

Primary cutaneous melanoma develops from an abnormal proliferation of melanocytes. The mechanism(s) that trigger and control this abnormal proliferation are not fully understood. Many other malignancies display recurring sites of chromosome changes (3,23,25,28,30,36). These nonrandom chromosomal abnormalities are useful in localizing and identifying genes that are central to the process of malignant transformation. Several cytogenetic studies of malignant melanoma, performed on advanced lesions (mainly metastasis) and established cell lines, have also reported nonrandom chromosomal abnormalities (8-11,14,29,32), Most of these studies reported a high level of aneuploidy in ceils, along with multiple rearrangements in chromosomes. The most frequently rearranged chromosomes found in these studies were chromosomes 1, 6, and 7. In the present investigation, ultrastructure and cytogenetic analy~es were performed in 10 cell lines derived from biopsies obtained from patients with metastatic melanoma. Our results show that the cell lines are mainly composed of melanoma cells with no fibroblasts, and karyotyping confirmed previous observations on the multiple chromosomal rearrangements involving chromosome 1, 6, and 7. However, in contrast to previous reports, we found a high frequency of changes involving chromosome 3. In 2 of the 10 cell