Mutant alleles of Schizosaccharomyces pombe rad9(+) alter hydroxyurea resistance, radioresistance and checkpoint control.

Schizosaccharomyces pombe rad9 mutations can render cells sensitive to hydroxyurea (HU), gamma-rays and UV light and eliminate associated checkpoint controls. In vitro mutagenesis was performed on S.pombe rad9 and altered alleles were transplaced into the genome to ascertain the functional significance of five groups of evolutionarily conserved amino acids. Most targeted regions were changed to alanines, whereas rad9-S3 encodes a protein devoid of 22 amino acids normally present in yeast but absent from mammalian Rad9 proteins. We examined whether these rad9 alleles confer radiation and HU sensitivity and whether the sensitivities correlate with checkpoint control deficiencies. One rad9 mutant allele was fully active, whereas four others demonstrated partial loss of function. rad9-S1, which contains alterations in a BH3-like domain, conferred HU resistance but increased sensitivity to gamma-rays and UV light, without affecting checkpoint controls. rad9-S2 reduced gamma-ray sensitivity marginally, without altering other phenotypes. Two alleles, rad9-S4 and rad9-S5, reduced HU sensitivity, radiosensitivity and caused aberrant checkpoint function. HU-induced checkpoint control could not be uncoupled from drug resistance. These results establish unique as well as overlapping functional domains within Rad9p and provide evidence that requirements of the protein for promoting resistance to radiation and HU are not identical.

Possible role of the receptor protein tyrosine phosphatase HmLAR2 in interbranch repulsion in a leech embryonic cell.

Accumulating evidence indicates that receptor protein tyrosine phosphatases (rPTPs) play major roles in growth cone migration. We have previously shown that the growth cones of the multiple parallel processes of an identified leech embryonic cell, the Comb cell (CC), express high levels of a leukocyte antigen-related (LAR)-like rPTP, HmLAR2. Embryonic injection of a polyclonal antibody to the receptor's ectodomain resulted in reduced process outgrowth and in processes crossing over each other, a behavior that is seldom observed in normal or control animals. Here we present results of injecting a soluble Fc-HmLAR2 ectodomain fusion protein into embryos in order to bind the endogenous ligands of HmLAR2. Single injections of the Fc-chimeric protein into the developing embryo resulted, 12 to 24 h postinjection, in clear morphological abnormalities, ranging from abnormally directed CC processes and crossovers to apparent growth cone collapse. At later times, 2 to 5 days post injection, growth cones appeared to have recovered and processes had continued to extend, but effects of the earlier guidance errors remained, with the CCs displaying a relatively high incidence of proximal guidance errors. When injected into the germinal plate of developing embryos, the fusion protein was found to bind selectively to the processes of the CCs themselves, in contrast to control injections of Fc alone or closely related Fc-tagged proteins, which did not decorate the CCs. Double-labeling experiments revealed an early phase of Fc-HmLAR2 labeling (within 20 min after application), during which the growth cones and filopodia of the CC showed significant binding of the receptor ectodomain, and a later phase (1-2 h after injection), when most of the label was redistributed away from the growth cones and into the proximal processes of the CC. In culture, HmLAR2-transfected COS cells were found to selectively bind the Fc-recombinant protein, but not Fc-tagged proteins bearing other closely related receptor ectodomains, demonstrating that the HmLAR2 ectodomain is capable of interacting homophilically. Together, our observations demonstrate that the rPTP HmLAR2 is critically involved in CC process extension through its participation in the regulation of growth cone structure, migration, and navigation. Moreover, since our experiments also indicate that HmLAR2 can bind to itself, we hypothesize that HmLAR2 has a key role in the mechanism of mutual repulsion that maintains the parallel growth of adjacent CC projections.

Molecular cloning and tissue-specific expression of Mrad9, a murine orthologue of the Schizosaccharomyces pombe rad9+ checkpoint control gene.

We have isolated a murine cDNA, Mrad9, that is orthologous to the fission yeast rad9+ and human HRAD9 genes. Mrad9 encodes a 389 amino acid long, 42,032 Dalton protein that is 27% identical and 56% similar to Rad9p, and 82% identical and 88% similar to HRAD9, at the amino acid level. Expression of the Mrad9 cDNA in Schizosaccharomyces pombe rad9::ura4+ cells restores nearly wild-type levels of hydroxyurea resistance and early S phase checkpoint control to mutant fission yeast cell populations. However, UV resistance is only minimally restored, and mutant cells remain sensitive to gamma radiation. Mrad9 genomic DNA was isolated from a mouse 129/SvEv library. The Mrad9 gene was local ized to a 15-kbp genomic DNA fragment, and contains 10 exons separated by 9 introns. Northern blot analysis indicates that the gene is expressed in many different tissues of the adult mouse, but the mRNA is most abundant in the heart and present at very low levels in the liver. These studies demonstrate the existence of a murine orthologue of the fission yeast rad9+ gene and underscore at least the partial evolutionary conservation of rad9+-dependent checkpoint control mechanisms.

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.

Inhibition of growth and induction of differentiation of metastatic melanoma cells in vitro by genistein: chemosensitivity is regulated by cellular p53.

We have investigated the effect of the soybean isoflavone genistein on the growth and differentiation of human melanoma cells. Four human melanoma cell lines, either completely lacking or containing different levels of wild-type p53, were treated with genistein in vitro in culture. It has been found that genistein significantly inhibited cell growth and that the chemosensitivity might depend on cellular p53 content. Specifically, the data suggest that high levels of wild-type p53 expression make cells resistant to genistein's growth-inhibitory action. Further support for this observation came from the stable transfection studies in which p53 transfectants expressing high levels of wild-type p53 became resistant to genistein. With respect to cell differentiation, our study showed that genistein increased melanin content and tyrosinase activity and caused the cells to form dendrite-like structures. Cells lacking p53 responded more than cells with p53 to dendrite-like structure formation. We also observed that genistein-induced differentiation involved an increase in tyrosinase mRNA level; the mechanisms by which genistein increases tyrosinase transcripts remain to be elucidated. Genistein treatment of the melanoma cell lines resulted in cell cycle arrest at G2/M check point and no significant apoptosis was observed.

Overexpression of EGF receptor is associated with spontaneous metastases of a human melanoma cell line in nude mice.

Twelve human melanoma cell lines were analyzed for alterations in the epidermal growth factor receptor (EGFR) gene at the DNA, RNA and protein levels. EGFR expression of the cell lines was then correlated with their previously reported p53 expression, in vivo growth characteristics, and rate of metastases in athymic mice. Northern blot and immunocytochemical analyses demonstrated low to intermediate levels of EGF receptor expression in four cell lines. Overexpression of EGFR was seen in one cell line, UISO-MEL-6. Although no significant statistical difference was observed between in vivo growth of EGFR-positive cell lines versus EGFR-negative cell lines, UISO MEL-6 which also lacked p53 expression, had the fastest in vivo rate of growth and was the only cell line to produce visceral metastases following subcutaneous inoculation in nude mice. Furthermore, EGFR overexpression in UISO-MEL-6 was associated with alterations of the gene at the DNA level.

Tumor suppressor p53 down-regulates tissue-specific expression of tyrosinase gene in human melanoma cell lines.

Tyrosinase, the key gene in melanin pigment synthesis, is tissue-specifically expressed in melanocytic cells. Expression of this gene is regulated by various hormones, carcinogens, and environmental factors. The molecular basis underlying tyrosinase gene regulation is still not clear. In this report, we present the effects of tumor suppressor p53 protein in tyrosinase gene expression and melanin synthesis in human melanoma. After stable transfection of wild type p53 expression plasmid into a highly pigmented melanoma cell line, overexpression of wt p53 suppressed the pigmentation of the melanoma cells. The loss of pigmentation was associated with the loss of endogenous tyrosinase expression at the activity and mRNA levels. In order to determine whether the p53 repression of tyrosinase mRNA involved modulation of tyrosinase promoter activity, transient transfection approaches involving p53 expression plasmid and construct containing chloramphenicol acetyl transferase (CAT) reporter gene linked to 270 bp tissue-specific tyrosinase promoter have been used. p53 specifically repressed CAT gene expression from the tyrosinase promoter and not from the Rous sarcoma virus promoter. These data suggest that in human melanoma p53 down-regulates the tissue-specific expression of tyrosinase gene and subsequent melanin synthesis.

Detection of dihydrotestosterone (DHT) doping: alterations in the steroid profile and reference ranges for DHT and its 5 alpha-metabolites.

Dihydrotestosterone (DHT), a biologically active metabolite of testosterone, may be misused in sports to benefit from its anabolic and psychotropic effects. After DHT application, a significant increase of the glucuronides of DHT and its metabolites can be expected for a certain time period depending upon dose, formulation, route of administration, and in case of percutaneous administration the chainlength of the ester. DHT and its metabolites can be monitored by gas-chromatography/mass spectrometry (GC/MS) after enzymatic hydrolysis and trimethylsilylation. To investigate the extent of the alteration of the urinary steroid profile after DHT application, timely controlled experiments have been performed with: a) oral application of [16,16,17-2H3]-DHT, and b) sublingual application of a 25 mg dose of DHT. In the experiment with [16,16,17-2H3]-DHT within 24 hours about 44% of the applied dose was recovered after hydrolysis with beta-glucuronidase from E. coli as di- or tri-deuterated 5 alpha-androstane glucuronides: androsterone (33.2%), 5 alpha-androsta-ne-3 alpha,17 beta-diol (2.5%), 5 alpha-androstane-3 beta, 17 beta-diol (0.9%), DHT (7.2%). Hydrolysis with beta-glucuronidase/arylsulfatase from Helix Pomatia resulted in a about 10% higher yield except for DHT. In the study with sublingual application of 25 mg of DHT the extent of the recovery of DHT and its metabolites was in the same range as for the deuterated DHT. The urinary glucuronide concentrations of DHT, androsterone (AND), 5 alpha-androstane-3 alpha, 17 beta-diol (5 alpha A3 alpha D) and 5 alpha-androstane-3 beta,17 beta-diol (5 alpha A3 beta D) and their ratios to etiocholanolone (ETIO), 5 beta-androstane-3 alpha, 17 beta-diol (5 beta A3 alpha D) and epitestosterone (EPIT) were increased for up to 48 hours after application. For doping control purposes concentrations of DHT, 5 alpha A3 alpha D, 5 alpha A3 beta D and ratios of 5 alpha-metabolites to non 5 alpha-metabolites such as DHT/ETIO, DHT/EPIT, 5 alpha A3 alpha D/5 beta A3 alpha D, 5 alpha A3 beta D/5 beta A3 alpha D, and AND/ETIO outside the reference ranges are a proof for DHT application. Reference ranges for Asian and Caucasian male and female athletes are calculated from data bases of the Asian Games 1994, the previous Asian Games 1990 and the routine doping control samples of Caucasian athletes measured in Cologne 1994. At the occasion of the 1994 Asian Games in Hiroshima alterations in the concentrations and ratios of the DHT depending parameters for outside there reference ranges have been found and have been sanctioned on this basis by the Medical Commission of the Organisation of Olympic Council of Asia (OCA).

Establishment of a human melanoma cell line lacking p53 expression and spontaneously metastasizing in nude mice.

Nine human melanoma cell lines established in our laboratory were analyzed for p53 gene expression and their tumorigenic and metastatic potential in nude mice. Northern blot analyses showed that five of the cell lines (55%) had either complete loss or low levels of p53 transcripts. Immunocytochemical analysis for p53 protein expression agreed with mRNA analysis results. Nucleotide sequencing showed no mutations in exons 5 through 8 of the gene. All cell lines except one gave rise to tumors at subcutaneous inoculation sites in nude mice. The melanoma cell line UISO-MEL-6, completely lacking p53 expression, spontaneously metastasized to lung and liver in nude mice.

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.

5-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 microM. 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 microM to 300 microM, 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 the ADA gene promoter to support diverse tissue-specific gene expression was investigated. Endogenous ADA gene expression in different mouse tissues was found to vary over a greater than 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-specific ADA expression was controlled by cis-acting sequences upstream of the coding region, constructs containing a reporter gene regulated by the ADA gene's 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.

Transfection and homologous recombination involving single-stranded DNA substrates in mammalian cells and nuclear extracts.

We have examined the ability of single-stranded DNA to participate in homologous recombination reactions in mammalian cells and nuclear extracts derived from them. We have inserted a fragment of the neo gene into the single-stranded DNA phage vector M13 mp11. The neo fragment was derived from a deletion derivative of the prokaryotic-eukaryotic shuttle vector pSV2neo. The resulting single-stranded DNA was mixed with a double-stranded deletion derivative of pSV2neo and tested for recombination in human cells, monkey cells, and nuclear extracts obtained from human cells. We were able to obtain recombinant molecules containing wild-type neo genes in all three systems. Examination of the products of recombination indicated that they resulted from correction of the deletion in the double-stranded DNA substrate. We were unable to detect any extensive conversion of single-stranded DNA into its double-stranded counterpart before it participated in the recombination reaction. We have also tested the ability of single-stranded DNA to yield transfectants. When a single-stranded DNA derivative of the herpes simplex virus thymidine kinase (TK) gene was introduced into mouse L-M(TK-) cells, we were able to obtain TK+ colonies. From these results, we conclude that single-stranded DNA can participate in transfection as well as homologous recombination reactions in mammalian cells.

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.