v-erbB related sequences in Xiphophorus that map to melanoma determining Mendelian loci and overexpress in a melanoma cell line

Certain genotypes of Xiphophorus harbour an accessory Mendelian gene which following loss, impairment, or insufficiency of its regulatory genes in the germ line, mediates the hereditary capacity to develop neoplasia spontaneously or following induction with initiating and promoting carcinogens. Together with its linked regulatory genes it forms a 'tumor gene-complex' (Tu-complex). We concentrated on accessory sex chromosomal Tu-complexes that are responsible for sex chromosome-linked melanoma formation. Southern analyses of the xiphophorine genome with 15 authentic oncogene probes revealed so far that only three v-erbB related EcoRI fragments comprising 4.9 kb of a certain X-, 11.5 kb of another X-, and 6.7 kb of both a Y- and Z-chromosome are inherited in parallel with the Tu-complex and melanoma formation. They are accessory in the genome, and are highly homologous with each other. The sequence of the X-chromosomal 4.9 kb fragment shows minor but significant differences to that of the invariably present autosomal xiphophorine erbB (x-erbB) fragment of 5.5 kb, indicating that at least two different x-erbB genes coding for different EGF receptors can exist in the fish. Northern analyses showed expression of both genes in a fibroblast cell line, and overexpression of the sex chromosomal x-erbB in a melanoma cell line. The co-segregation of the hereditary trait of melanoma with the sex chromosomal x-erbB fragments, suggests that the accessory x-erbB gene may be responsible for the switch from the normal to the neoplastic state of the pigment cells.     

Mechanisms underlying resistance to cetuximab in the HNSCC cell line: role of AKT inhibition in bypassing this resistance

EGFR is frequently overexpressed in head and neck squamous cell cancer (HNSCC). Cetuximab is a monoclonal antibody designed to interact with EGFR, block its activation, reduce the downstream signaling pathways and induce EGFR internalization. This study aims to investigate the role of the EGFR signaling pathway and EGFR internalization in a cetuximab-resistant cell line and to propose a new therapeutic strategy to optimize treatment of HNSCC. The HNSCC cell line, CAL33 was sensitive to gefitinib but resistant to cetuximab. Cetuximab induces an unexpected EGFR phosphorylation in CAL33 cells similarly to EGF but this EGFR activation does not trigger EGFR internalization/degradation, the process currently implicated in the response to cetuximab. Cetuximab inhibits ERK and AKT phosphorylation in cetuximab-sensitive A431 cells, whereas the level of AKT phosphorylation is unmodified in cetuximab-resistant cells. Interestingly, CAL33 cells harbor a PIK3CA mutation. The treatment of CAL33 cells with PI3K inhibitor and cetuximab restores the inhibition of AKT phosphorylation and induces growth inhibition. Our results indicate that EGFR internalization is impaired by cetuximab treatment in CAL33 cells and that the AKT pathway is a central element in cetuximab resistance. The combination of cetuximab with a PI3K inhibitor could be a good therapeutic option in PIK3CA-mutated HNSCC.     

DNA methylating capacity in metastatic variants of a human melanoma cell line

Certain highly metastatic (in athymic immunosuppressed "nude" mice) variants of the poorly metastatic human melanoma cell line MeWo have been found to contain dramatically reduced levels of DNA 5-methylcytosine compared to the parental cell line. To identify the underlying biochemical defect which could be responsible for the reduced DNA methylation within these cells, the intracellular ratio of S-adenosylmethionine/S-adenosylhomocysteine and level of extractable DNA-methyltransferase were examined. No significant difference in the ratio of S-adenosylmethionine/S-adenosylhomocysteine or extractable DNA-methyltransferase activity were found between the highly malignant variants and the parental cell line. Thus, stable alterations in these cellular parameters are not likely responsible for the reduction in DNA 5-methylcytosine content which appears to occur during "progression" of the MeWo tumor line from a relatively benign to highly malignant state.     

Characterization of a human melanoma cell line with non-oestrogen receptor dependent tamoxifen resistance

While investigating the mechanism of synergistic cytotoxicity between tamoxifen (TAM) and cisplatin (DDP) we developed a TAM-resistant variant of the human melanoma cell line T-289. We sought to characterize this cell line with respect to the effect of TAM resistance on a variety of different intracellular cell cycle control and apoptotic pathways. A TAM-resistant variant cell line (289 TAMs) was developed and the technique of Western analysis was to determine the changes in protein expression that occurred as a result of the development of TAM resistance. In this model, TAM resistance resulted in an increase in the detectable basal levels of cyclin E, GADD 153, p16, BAX, Bcl-XL, and wild-type and mutant p53, an increase in TAM induction of p16, and a decrease in the detectable basal levels of cyclin D1, p21 and p27. There were no detectable changes in the levels of pRb. In the TAM-resistant variant, p21 levels were essentially undetectable, while p27 was present and maintained its response to TAM Induction, albeit at a much lower level. This investigation demonstrates that the development of TAM resistance is associated with a change in the detectable levels of a variety of cell cycle control and apoptosis-related proteins. While the exact role that each change plays in the development of tamoxifen resistance remains to be determined, these data suggest that the development of resistance to a particular agent results in changes in multiple, seemingly unrelated pathways. These data have implications for future studies in both the laboratory and the clinic.     

Mechanisms of resistance to cis-diamminedichloroplatinum (II) in a rat ovarian carcinoma cell line

A cis-diamminedichloroplatinum(II) (cisplatin)-resistant subline (Cis-Pt^r) demonstrated 20-fold greater resistance to the cytotoxic effects of cisplatin, compared with the parental cloned rat ovarian carcinoma cell line (ROT 68/C1). The uptake of cisplatin into the Cis-Pt^r cells was identical to that into the ROT68/C1 cells in vitro and in vivo. Glutathione activity in a cytoplasmic extract was 1.4-fold and 1.8-fold greater in the Cis-PT^r cells than in the ROT68/C1 cells in vitro and in vivo, respectively. There was no difference between the ROT68/C1 and Cis-PT^r cells in ^195mcisplatin binding per μg DNA. DNA repair of cisplatin DNA damage was increased in the Cis-PT^r cells but not in the ROT68/C1 cells. These results suggest that the mechanism of resistance to cisplatin in rat ovarian carcinoma cells involve increased activity of the DNA repair system and increased cytosolic binding to thiols may also be involved.     

Amplified KpnL repetitive DNA sequences in homogeneously staining regions of a human melanoma cell line

The human melanoma cell line MeWo was found to contain two populations of cells, one containing 83 chromosomes (hypotetraploid) and the other containing 43 chromosomes (hypodiploid). All of the hypodiploid cells, but none of the hypotetraploid cells, contained chromosomes with long homogeneously staining regions (HSR's) when examined with quinacrine fluorescence. These long HSR's on an X-chromosome and derivative chromosome 15, produced characteristic patterns of positive- and negative-staining areas along the HSR's with both conventional Giemsa (G) staining and C-banding. The C- and G-positive regions stained with distamycin A-DAPI, which is specific for the centromeric heterochromatin of chromosomes 1, 9, 15p, 16, and Y. DNA extracted from MeWo cells and digested with the restriction enzymes KpnL or Sau3A exhibited marked amplification of a 1.8-kilobase fragment. The amplified Sau3A fragment (D15Z1) was cloned, mapped, and partially sequenced. The sequenced region contained a five-base-pair repeat unit (adenine-adenine-thymine-guanine-guanine) that has undergone considerable divergence. Estimates of the size of the HSR's and the amount of amplified DNA suggested that each HSR contained at least 30,000 copies of the 1.8-kb KpnL fragment, representing about 50% of each HSR. The amplified sequence was identified as one member of the previously described KpnL family of repeated sequences. In situ hybridization of D15Z1 to MeWo metaphase chromosomes resulted in heavy labeling over both HSR's. These data suggested that centromeric heterochromatin and neighboring sequences on chromosome 15 have been amplified and some of this material translocated to the X-chromosome.     

Doxorubicin cellular pharmacokinetics and DNA breakage in a multi-drug resistant B16 melanoma cell line

Mechanisms of anthracycline resistance have been investigated in a B16 murine melanoma cell subline selected by continuous in vitro exposure to increasing concentrations of doxorubicin (DX). Altered drug pharmacokinetics were observed in resistant B16 cells as compared to the sensitive counterpart. In fact, cellular DX uptake - as determined by a fluorescence method - was lower in resistant than in sensitive cells. Furthermore, drug efflux rate was shown to be higher in resistant than in sensitive cells; treatment of cells with the metabolic inhibitor sodium azide decreased drug efflux rate in resistant but not in sensitive cells, suggesting the presence of an energy-dependent drug extrusion mechanism in the resistant B16 cells. However, since drug-induced cell killing did not correlate with cellular DX contents in sensitive and resistant cells, drug resistance of B16 subline could not be completely explained by the observed differences in drug pharmacokinetics. Since drug-induced DNA breaks have been related to drug cytotoxicity, DNA cleavage was also measured by alkaline elution methods. The number of DNA breaks produced by DX was decreased in resistant cells as compared to sensitive cells at the same cellular drug accumulation. The results are consistent with the view that anthracycline resistance may be multifactorial and probably arises following multiple biochemical changes.     

DNA amplification and metastasis of the human melanoma cell line MeWo

A positive correlation was found between the presence of amplified DNA in the form of homogeneously staining regions (HSRs), and the formation of spontaneous metastases by the human melanoma cell line MeWo. HSR+ and HSR- MeWo sublines and clones were injected s.c. into BALB/c nude mice. All MeWo lines produced primary tumors that were allowed to grow to a similar size before the animals were sacrificed and examined for the presence of metastatic nodules in the lungs and abdominal cavity. HSR+ lines gave extensive metastases (greater than 100 nodules) in the lungs and/or liver and abdomen in 18 of 19 animals. The HSR- MeWo lines were effectively nonmetastatic, producing metastases in 3 of 20 animals, two of which had only a single metastatic lung nodule each. Evidence for the presence of HSRs in the primary tumors and metastatic nodules was obtained by DNA dot-blot hybridization to a sequence (D15Z1) amplified in the HSRs, flow cytofluorometry for cellular DNA content, and quinacrine staining of metaphase spreads. The HSR+ clones also colonized the lung to a much greater extent than HSR- clones following i.v. injection. In addition, the HSR+ clone had a selective advantage in lung colonization, since i.v. injection of a 50:50 mixture of HSR+ and HSR- clones resulted in extensive metastases populated exclusively by HSR+ cells. The results suggest that DNA sequences amplified in the HSRs of human melanoma MeWo cells may confer enhanced metastatic properties to these cells.     

DNA amplification and tumorigenicity of the human melanoma cell line MeWo

Homogeneously staining regions (HSRs) were found in hypodiploid cells (40%) of a subline of the human melanoma cell line, MeWo, (MeWo-C) but were absent from the hypotetraploid cells (60%). Another subline (MeWo-B) was also shown to contain two populations of cells, 70% hypodiploid and 30% hypotetraploid. None of the MeWo-B cells contained HSRs, but all four cell types from both sublines shared marker chromosomes indicating their common origin. The hypodiploid MeWo-B cells were karyotypically similar to the hypodiploid MeWo-C cells except for the presence of the HSRs in the latter. Both MeWo-C and MeWo-B sublines were injected into BALB/c nude mice. The MeWo-C cells were markedly more tumorigenic than MeWo-B cells as judged by tumor incidence, latency, average tumor size, and tumor take values. Cytogenetic and flow cytofluorometric analyses of the tumors induced by MeWo-C cells revealed a shift in the tumor cell population from 40% to greater than 90% HSR-containing hypodiploid cells during tumor growth. Hybridization of tumor DNA to a probe (D15Z1), the sequence of which is amplified in the HSRs, also indicated an increase in the proportion of HSR-bearing cells during tumor growth. No such selective advantage was found with the hypodiploid, HSR-lacking MeWo-B cells. The results suggest that HSRs found in the human melanoma line MeWo may confer enhanced tumorigenicity to the cells containing them.     

High frequency of double drug resistance in the B16 melanoma cell line

Methotrexate (MTX) and N-(phosphonacetyl)-l-aspartate (PALA) are two agents to which cellular resistance can be conferred by gene amplification, but they do not generally show cross resistance. However, combined treatment with these two agents produced drug resistant cells in the B16 melanoma cell line at a much higher frequency than would be expected if resistance to the two agents was totally independent. An isolated doubly resistant clone, B16-F1 MP, showed a high frequency of resistance to pyrazofurin and ouabain, which are also agents to which resistance can be conferred by gene amplification. Thus MTX combined with PALA selected cells with an ‘amplificator’ phenotype (an increased ability to amplify parts of the genome). These B16-F1 MP cells had a decreased ability to form experimental lung metastases compared with the parent line but this difference was not found in baby hamster kidney cells with the amplificator phenotype. The mechanism underlying drug resistance may need to be considered when designing combination chemotherapy.     

Genomic stability in malignant melanoma of Xiphophorus

Microsatellite instability is a feature of many tumours and is indicative of a generalized genomic instability of cancer cells. Whether this phenomenon is essential for tumorigenesis and whether it is an early or late step is still a matter of debate. In the Xiphophorus melanoma model, the primary steps leading to tumour formation are known and include overexpression of a mutationally altered epidermal growth factor receptor and the resulting defects in signalling. We have analysed the late stages of melanoma progression for microsatellite instability. Although several types of microsatellite allele alteration in DNA from tumours relative to DNA from non-tumour tissue were found, the frequency was rather low (7.6%). Thus, although the tumours show a wide range of malignancy and aggressiveness, genomic instability that becomes apparent as microsatellite instability does not appear to be an obligatory step for melanoma progression.     

Testosterone augments polyphenol-induced DNA damage response in prostate cancer cell line, LNCaP

Recently, we reported that combined ingestion of soy isoflavones and curcumin significantly decreased the serum level of prostate-specific antigen based on a randomized placebo-controlled double-blind clinical study. We investigated whether these polyphenols inhibited the proliferation of prostate cancer cells by activating a DNA damage response. The effects of isoflavones and curcumin on the expression and phosphorylation of ataxia-telangiectasia-mutated kinase (ATM), histone H2AX variant (H2AX) and checkpoint kinase2 (Chk2) were examined in LNCaP cells. The induction of apoptosis in LNCaP cells was evaluated by poly(ADP-ribose) polymerase (PARP) cleavage. Furthermore, the effects of a testosterone supplement on modulation of the DNA damage response were examined. Combined treatment of isoflavones and curcumin additively suppressed cellular proliferation and induced phosphorylation of ATM, histone H2AX, Chk2 and p53. Testosterone augmented the activation of the DNA damage response and PARP cleavage induced by curcumin. Our results indicate that activation of the DNA damage response by polyphenols might suppress the malignant transformation of prostate cancer. In addition, testosterone, when combined with curcumin, may have suppressive effects on the progression of prostate cancer.     

Non-P-glycoprotein-mediated multidrug resistance in a small cell lung cancer cell line: evidence for decreased susceptibility to drug-induced DNA damage and reduced levels of topoisomerase II.

Data obtained from clinical samples suggest that non-P-glycoprotein mechanisms of multidrug resistance are likely to be important in small cell lung cancer. The H69AR cell line was derived from the H69 small cell lung cancer cell line by selection in doxorubicin (adriamycin) and does not overexpress P-glycoprotein as detected by monoclonal antibody C219 (S.E.L. Mirski et al., Cancer Res., 47:2594, 1987). In the present study, we have used the polymerase chain reaction to verify that H69AR cells do not overexpress P-glycoprotein. Further, transport studies with radiolabeled daunomycin, VP-16, and vinblastine demonstrate that differences in net drug accumulation or efflux are not part of the resistance phenotype of H69AR cells. To determine if H69 and H69AR cells differ in their susceptibility to drug-induced DNA damage, DNA single-strand breaks (SSB) generated by VP-16 and Adriamycin were measured using the alkaline filter elution assay. Readily detectable SSB were produced in intact H69 cells by 5 microM VP-16, but 100 microM drug was required to cause similar damage in H69AR cells. H69AR cells were also resistant to SSB induction by Adriamycin. The formation of SSB by VP-16 was similarly reduced in isolated H69AR nuclei, indicating that resistance to this drug resides, at least in part, in the nucleus. No significant differences were observed in the rate or extent of repair of VP-16-induced DNA SSB in H69 and H69AR cells. The reduced susceptibility to drug-induced SSB may result from alterations in topoisomerase II, since less immunoreactive topoisomerase II was found in H69AR cells compared to H69 cells. However, changes in topoisomerase II cannot explain the resistance of H69AR cells to such drugs as the Vinca alkaloids and gramicidin D, indicating that multiple mechanisms contribute to drug resistance in this small cell lung cancer cell line.     

Isolation and characterization of a breast progenitor epithelial cell line with robust DNA damage responses

Summary We report the establishment of a breast epithelial cell model that undergoes growth arrest at different stages of the cell cycle depending upon the DNA damaging agents encountered. Primary breast epithelial cells from normal reductive mammoplasty were grown in low-calcium culture medium. Free-floating cells under this condition were separated and used for establishment of the MCF-15 breast epithelial cell line. We found that MCF-15 breast epithelial cells display a superb response to different phases of the cell cycle arrest in response to various DNA damaging agents. Immunohistological analysis indicates that MCF-15 cells express cytokeratin 19, CD44, CXCR4, SDF-1, SPARC and vimentin. Although less than 5% of the MCF-15 cells expressed Muc-1 in culture, increased Muc-1 expression was observed in luminal epithelial cells along the newly formed lumen in xenografts. Furthermore, a small population of MCF-15 cells expressed estrogen receptor-α (ERα) in xenografts while ERα expression was not detected in monolayer culture. Therefore, the MCF-15 breast epithelial cell line possesses characteristics of breast progenitor cells and provides a good cell culture model for studying the response to DNA damage and the etiology of aggressive basal-like breast cancers.     

Metastasis of a human melanoma cell line in the nude mouse

A human melanoma cell line has been established which when inoculated subcutaneously into nude mice, is consistently metastatic. In order to document blood-borne spread, it was necessary to excise the primary tumour so prolonging the life of the animal and allowing metastases to become apparent. Macroscopically detectable metastatic spread at autopsy was reliably indicated by weight loss of the animals. Metastases were widespread and involved the lungs, abdominal cavity and organs and the gonads. The size of the primary tumour at the time of its removal, and not the period of s.c. growth, determined the incidence of metastatic disease. Removal of tumours weighing less than 0.6 g prevented metastasis, whereas all of the animals showed widely disseminated disease if the tumour was allowed to attain a size of 1.6 g before excision.     

Stimulation of melanogenesis in a human melanoma cell line by retinoids

Retinoic acid was found to be a potent stimulant of pigmentation in human Hs939 melanoma cells. Exposure to 1 microM retinoic acid for longer than four days caused both a decrease in the rate of cell proliferation and a concomitant increase in melanogenesis. These effects of retinoic acid progressed lin-early in a time-dependent and a dose-dependent fashion such that at the end of a seven-day treatment cell growth was inhibited by approximately 65%, and both melanin content and tyrosinase activity increased more than three-fold over the control. Interpolation of the dose-response curves indicated that 3 nM retinoic acid would cause half-maximal melanogenesis stimulation. No elevation in the level of cyclic adenosine 3':5'-monophosphate could be detected in the melanoma cells following various periods of exposure to retinoic acid, and the cells were unresponsive to alpha-melanocyte-stimulating hormone. In the presence of the tyrosinase inhibitor phenylthiocarbamate, retinoic acid was capable of inhibiting cell proliferation without enhancing melanin synthesis. The tumor promoter phorbol myristate acetate did not affect either the proliferation or the differentiation of the Hs939 melanoma cells. However, the enhancement of melanogenesis by 1 microM retinoic acid was inhibited by 66% in the presence of 0.1 microM phorbol myristate acetate. The tumor promoter did not reverse the growth-inhibitory effect of retinoic acid. Phorbol, a non-tumor promoter, was effective. Other retinoids, such as 13-cis-retinoic acid, retinyl acetate, nd the trimethylmethoxyphenyl analog of retinoic acid, also inhibited the proliferation and enhanced melanin production in the Hs939 cells. In contrast, retinyl palmitate, the phenyl analog of retinoic acid, and the pyridyl analog of retinoic acid were ineffective.     

Characterization of a cathepsin-H-like enzyme from a human melanoma cell line

A cathepsin-H-like enzyme has been isolated from cultured human melanoma cells (G 361 cell line). The enzyme is similar to cathepsin H(s) of normal tissues in molecular weight, enzymatic characteristics (substrates, inhibitors, pH optima, Km values), and immunoreactivity. The inactive form of the enzyme with a molecular mass of 40 kDa has been found in the culture medium. The inactive enzyme is activated by acid pH, pepsin, and cathepsin-D-like enzyme treatments and converted into a form with a molecular mass of 28 kDa. The activated extracellular cathepsin-H-like enzyme and the active intracellular enzyme exhibit the same characteristics. The melanoma-derived cathepsin-H-like enzyme degrade fibrinogen and fibronectin, but not laminin or type-IV collagen. We conclude that the extracellular cathepsin-H-like enzyme may have important functions, together with other proteinases, in the destruction of extracellular matrix components, thus enabling proliferation, migration, and metastasis to occur.     

Mapping of tyrosine kinase gene family members in a Xiphophorus melanoma model

Xiphophorus fish have been the subject of intensive genetic research for more than 60 yr, primarily because of the availability of a number of interspecific hybrids that are malignant melanoma models with apparently simple oncogene and tumor suppressor gene determinants. The gene map of Xiphophorus is one of the most extensive among nonhuman vertebrates, with about 100 genes assigned to at least 20 independently assorting linkage groups (LGs), as well as more than 250 anonymous DNA sequence markers, providing coverage for most of the genome for genetic mapping studies. This characteristic has resulted in the mapping of a tumor suppressor locus, DIFF, which is one of two genetic determinants of melanoma formation in the best-studied hybrid melanoma, the Gordon-Kosswig melanoma model. The other gene responsible for melanoma formation in this model is a sex-linked tyrosine kinase gene related to EGFR and called Xiphophorus melanoma receptor kinase (Xmrk). The cellular oncogene homologues of the non-receptor tyrosine kinase family orthologous to yes and fyn have also been found to be overexpressed in malignant melanomas of Xiphophorus and may be involved in tumor progression. We report here the map location of a Xiphophorus yes gene, YES1, in LG VI, closest to the EGFR gene and the assignment of a fyn gene homologue to newly designated LG XV, linked to the gene for cytosolic α-galactosidase. We also confirmed that an EGFR-related sequence (EGFRL1) that we previously assigned to Xiphophorus LG VI by cross-hybridization to a viral erbB probe was the EGFR orthologue. Our results suggest that the presence of expressed duplicates of members of the tyrosine kinase gene family in teleost fishes may increase the potential number of targets in oncogenic cascades in fish tumor models. Mol. Carcinog. 22:150–157, 1998. © 1998 Wiley-Liss, Inc.