Etiology of MNU-induced melanomas in Xiphophorus hybrids.

Genetic hybrids of the genus Xiphophorus have historically been useful models for study of the genetic aspects of tumor formation. In the most studied Xiphophorus tumor model, two-gene loci, XMRK and DIFF, are implicated as critical both to UV-induced and spontaneous melanoma formation in BC(1) hybrids of crosses between X. maculatus and X. helleri, with X. helleri as the recurrent backcross parent. In addition to UV, the direct-acting carcinogen N-methyl-N-nitrosourea (MNU) has been used to induce tumors in Xiphophorus BC(1) hybrids from several cross types. In the present study, we address the hypothesis that excess melanomas in MNU-treated BC(1) hybrids may have been generated by direct mutation of CDKN2AB, a candidate gene for DIFF. MNU treatment of F(1) and BC(1) hybrid fish significantly increased tumor incidence at 6 months; however, no association was found between MNU-induced tumor formation and zygosity of the candidate tumor tumor-suppressor CDKN2AB in BC(1) hybrids, consistent with previously reported results. Sequence analysis of the X. maculatus CDKN2AB locus of heterozygous individuals (both BC(1) and F(1) hybrids) did not reveal any mutations caused by MNU, suggesting that the mechanism of MNU-induced melanoma formation in this Xiphophorus model does not involve direct mutation of CDKN2AB but may result from mutation of other critical genes.

Melanoma susceptibility and cell cycle genes in Xiphophorus hybrids.

Xiphophorus interspecies hybrids provide genetically defined models of both spontaneous and inducible melanomagenesis. In both models, backcrossing F(1) hybrids of different strains of X. maculatus and X. helleri to a X. helleri parental fish results in segregation of melanoma susceptibility, fitting a Mendelian two-gene inheritance model. The sex-linked Xmrk oncogene is required for melanoma development in both crosses. The Xiphophorus CDKN2A/B gene, which is homologous to mammalian CDKN2A/B cyclin-dependent kinase inhibitors (p16 and p15), is a candidate melanoma susceptibility gene. In this model, tumor susceptibility segregates with homozgyosity for CDKN2A/B from the recurrent X. helleri parent in backcross hybrids. We found that both CDKN2A/B mRNA and protein are highly overexpressed in melanoma. Because the p13 protein product of CDKN2A/B is a putative regulator of the G1 checkpoint, we investigated expression of other components of Xiphophorus G1 checkpoint control. By real-time PCR analysis, retinoblastoma gene (RB) is consistently expressed twofold higher in both tumors and melanized skin than in normal tissue, indicating that RB is not downregulated by the overexpression of CDKN2A/B in Xiphophorus melanoma. We also found a significant correlation between the quantitative level of CDKN2A/B and Xmrk RNA in tumors, suggesting a functional relationship between Xmrk and CDKN2A/B expression. Although X. helleri CDKN2A/B protein contains a non-conservative substitution, the biochemical function appears to show little overt defect. These studies indicate that in Xiphophorus melanoma, CDKN2A/B is functionally insufficient to mediate cell-cycle arrest in the presence of Xmrk.

Regulation of CDKN2A/B and Retinoblastoma genes in Xiphophorus melanoma.

Xiphophorus interspecies hybrids provide several well-characterized genetic models of melanoma susceptibility. The Xiphophorus CDKN2A/B gene, homologous to mammalian CDKN2A/B cyclin-dependent kinase inhibitors (p16 and p15), is a candidate tumor susceptibility gene in these models. Using real-time PCR and Western blot analysis, we analyzed expression of CDKN2A/B in spontaneous and UV-induced primary melanomas from individual backcross hybrid fish. We found that CDKN2A/B mRNA is highly expressed in melanomas (18-fold), relative to other fish tissues. Expression is also elevated, to a lesser extent (9.5-fold), in melanized skin from tumor-bearing fish. However, quantitative levels of CDKN2A/B mRNA in tumors varied considerably and positively correlated with expression of the Xmrk oncogene, suggesting possible functional interaction between Xmrk and CDKN2A/B expression. As a homolog corresponding to members of the mammalian CDKN2 family which regulate cell cycle progression at the G1 checkpoint, the CDKN2A/B p13 protein is a putative regulator of the G1 checkpoint apparatus in Xiphophorus. Since CDKN2A is often observed to be inversely regulated compared to RB in some human tumors, and is capable of transcriptionally regulating RB in human ovarian tumors, we cloned the Xiphophorus maculatus RB cDNA and analyzed RB expression by real-time PCR and Western blot analysis in the fish melanomas. These experiments were designed to ascertain whether CDKN2A/B and RB expression were inversely correlated. Our results indicate that RB mRNA was consistently expressed at only a 2-fold higher level in both tumors and melanized skin than in muscle. Qualitatively similar results were obtained for protein expression. These results collectively suggest that (i) Xmrk and CDKN2A/B may be co-regulated at the transcriptional level, and (ii) there is little, if any, alteration of RB expression in Xiphophorus melanomas.

Induction of prosurvival molecules by apoptotic stimuli: involvement of FOXO3a and ROS.

Most cancer therapeutics fails to eradicate cancer because cancer cells rapidly develop resistance to its proapoptotic effects. The underlying mechanisms remain incompletely understood. Here we show that three representative apoptotic stimuli, that is, serum starvation, a mitochondrial toxin, and a DNA-damaging agent (etoposide), rapidly induce several distinct classes of prosurvival molecules, in particular, Bcl-2/Bcl-X(L) and superoxide dismutase (SOD; including both MnSOD and Cu/ZnSOD). At the population level, the induction of these prosurvival molecules occurs prior to or concomitant with the induction of proapoptotic molecules such as Bim and Bak. Blocking the induction using siRNAs of the prosurvival or proapoptotic molecules facilitates or inhibits apoptosis, respectively. One master transcription factor, FOXO3a, is involved in the transcriptional activation of some of these prosurvival (e.g., MnSOD) and proapoptotic (e.g., Bim) molecules. Interestingly, in all three apoptotic systems, FOXO3a itself is also upregulated at the transcriptional level. Mechanistic studies indicate that reactive oxygen species (ROS) are rapidly induced upon apoptotic stimulation and that ROS inhibitors/scavengers block the induction of FOXO3a, MnSOD, and Bim. Finally, we show that apoptotic stimuli also upregulate prosurvival molecules in normal diploid human fibroblasts and at subapoptotic concentrations. Taken together, these results suggest that various apoptotic inducers may rapidly mobilize prosurvival mechanisms through ROS-activated master transcription factors such as FOXO3a. The results imply that effective anticancer therapeutics may need to combine both apoptosis-inducing and survival-suppressing strategies.

Evidence that Sp1 positively and Sp3 negatively regulate and androgen does not directly regulate functional tumor suppressor 15-lipoxygenase 2 (15-LOX2) gene expression in normal human prostate epithelial cells.

In this project, we studied the gene regulation of 15-lipoxygenase 2 (15-LOX2), the most abundant arachidonate-metabolizing LOX in adult human prostate and a negative cell-cycle regulator in normal human prostate (NHP) epithelial cells. Through detailed in silico promoter examination and promoter deletion and activity analysis, we found that several Sp1 sites (i.e., three GC boxes and one CACCC box) in the proximal promoter region play a critical role in regulating 15-LOX2 expression in NHP cells. Several pieces of evidence further suggest that the Sp1 and Sp3 proteins play a physiologically important role in positively and negatively regulating the 15-LOX2 gene expression, respectively. First, mutations in the GC boxes affected the 15-LOX2 promoter activity. Second, both Sp1 and Sp3 proteins were detected in the protein complexes that bound the GC boxes revealed by electrophoretic mobility shift assay. Third, importantly, inhibition of Sp1 activity or overexpression of Sp3 both inhibited the endogenous 15-LOX2 mRNA expression. Since 15-LOX2 is normally expressed in the prostate luminal epithelial cells, we subsequently explored whether androgen/androgen receptor may directly regulate its gene expression. The results indicate that androgen does not directly regulate 15-LOX2 gene expression. Together, these observations provide insight on how 15-LOX2 gene expression may be regulated in NHP cells.

Cloning and analysis of a FoxO transcription factor from Xiphophorus.

Melanoma development in the fish Xiphophorus is determined, at least in part, by overexpression and activation of the Xmrk-2 oncogene, which triggers a variety of signal transduction pathways resulting in altered cell cycle control. We have begun analysing transcription factors which may link Xmrk-2 with regulation of cell proliferation or apoptosis. Towards this end, we have cloned an FKHR (FoxO sub-family) homolog from Xiphophorus maculatus. The isolated clone is a 2.7 kb cDNA encoding a predicted protein of 664 amino acids. The gene, which we have named FoxO5, maps to Xiphophorus Linkage Group XV. The protein product can be categorized within a branch of the FOXO sub-class, which includes: Danio rerio zFKHR (foxo5), Homo sapiens FKHR-L1 (FoxO3a) and Mus musculus FKHR2 (Foxo3). Notably, the Forkhead DNA binding domain, three Akt consensus phosphorylation sites and a carboxy-terminal minimal activation domain are each highly conserved. A mutated FoxO5 protein with disrupted Akt phosphorylation sites inhibits proliferation, but the wild-type protein fails to do so, when exogenously expressed in Xiphophorus cells derived from a melanoma. The same mutated protein predominantly localizes to the nucleus, yet the wild-type protein seldom does. Further characterization of Xiphophorus FoxO5 will contribute to understanding the molecular basis of carcinogenesis in these species.