Andrew P. Butler

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-XL 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.

Enhanced Sp1 DNA-binding activity in murine keratinocyte cell lines and epidermal tumors

Altered regulation of ornithine decarboxylase (ODC) is frequently observed in epidermal tumors. We have shown that the transcription factor Sp1 is one of the regulators of ODC expression and that Sp3 antagonizes this Sp1-mediated activation of ODC expression. These results led us to examine the levels and binding activity of Sp1 and Sp3 in nuclear extracts prepared from cultured murine keratinocytes, transformed keratinocyte cell lines and epidermal tumors. Here we show that the Sp1 DNA-binding activity is higher in established keratinocyte cell line extracts than in primary keratinocyte extracts. Sp1 message levels and Sp1 DNA-binding activity was found to be low in 20-week papillomas and high in squamous cell carcinomas. These results suggest that increased levels of Sp1 and enhanced Sp1 DNA binding activity are correlated with epidermal tumor progression. Based on these results, we propose that increased Sp1 DNA binding may augment the proliferative capacity of tumor cells through overexpression of Sp1-responsive genes, possibly including ODC.

Genetic analysis of susceptibility to spontaneous and UV-induced carcinogenesis in Xiphophorus hybrid fish.

Abstract:Xiphophorus interspecies hybrids provide genetically controlled models of tumor formation. Spontaneous melanomas form in first-generation backcross (BC1) hybrids produced from backcrossing F1 hybrids derived from the platyfish X. maculatus Jp 163 A and the swordtail X. helleri to the X. helleri parental strain (the Gordon-Kosswig hybrid cross). Nodular melanomas originate in the dorsal fin from cells constituting the spotted dorsal (Sd) pigment pattern. A parallel genetic cross, with X. maculatus Jp 163 B, exhibits the spotted side (Sp) pigment pattern instead of Sd, and produces BC1 hybrids exhibiting a much lower frequency of spontaneous melanoma formation. These hybrids are susceptible to melanoma development if irradiated with UV light as fry. Other hybrids involving these two strains of X. maculatus and different swordtail and platyfish backcross parents also have been investigated as potential tumor models, and show differing susceptibilities to UV-induced and spontaneous melanomas. Genotyping of individual BC1 hybrids from several Xiphophorus crosses has implicated a locus, CDKN2X (a Xiphophorus homologue of the mammalian CDKN2 gene family, residing on Xiphophorus linkage group V), in enhancing pigmentation and the susceptibility to spontaneous and UV-induced melanoma formation in BC1 hybrids from some crosses, but not others. Homozygosity for X. helleri and X. couchianusCDKN2X alleles in BC1 hybrids can predispose individuals to melanoma, but this susceptibility is modified in other crosses depending both on the contributing sex-linked pigment pattern locus from X. maculatus (Sd or Sp), and the genetic constitution of the backcross parent. Xiphophorus BC1 hybrids constitute unique genetic models offering the potential to analyze the contributions of specific genes to spontaneous and induced tumor formation in different, but comparable genetic backgrounds.

Involvement of protein kinase C in the regulation of ornithine decarboxylase mRNA by phorbol esters in rat hepatoma cells

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulates a rapid increase in ornithine decarboxylase (EC 4.1.1.17; ODC) activity in target cells. Here we demonstrate that this process involves a rapid accumulation of ODC mRNA, which is maximal 3 h after treatment (three- to eightfold greater than control cells) and decays to control levels within 18 h. Stimulation of ODC mRNA by TPA is blocked by phorbol dibutyrate down-regulation of protein kinase C (PKC). ODC mRNA was also induced by the PKC activators, phospholipase C and 1-oleoyl-2-acetyl-rac-glycerol, and blocked by kinase inhibitors (trifluoroperazine, H7, and palmitoyl-L-carnitine), consistent with a requirement for PKC activation in the induction mechanism. However, the non-PKC-specific protein kinase inhibitor HA1004 also suppressed expression of ODC mRNA in response to TPA, under conditions where it did not inhibit PKC, suggesting that additional kinases may be involved in the intracellular signalling process. The stability of the ODC mRNA (control value = 6.2 +/- 1.6 h) is not significantly changed by either TPA (5.7 +/- 0.8 h) or by cycloheximide (6.0 h). These results are inconsistent with any contribution from altered mRNA half-life towards the accumulation of ODC mRNA following treatment with phorbol ester tumor promoters.

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.

Core promoter involvement in the induction of rat ornithine decarboxylase by phorbol esters.

Overexpression of ornithine decarboxylase (ODC) is an important oncogenic event in tumorigenesis. Although ODC was one of the first genes described whose product is inducible by 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA), the mechanisms of ODC transcriptional regulation have remained elusive. In this study, we systematically analyzed the rat ODC core promoter region for novel TPA response elements. Analysis of linker scanning mutants of the ODC promoter from the TATA box to the transcription start site demonstrated that mutation of the TATA box reduced the TPA induction ratio by 40%, while the basal ODC promoter activity was not significantly changed. A novel region between nt − 20 to − 10 was shown to be critical for both basal promoter activity and induction by TPA. Random mutagenesis of this region showed that conversion of the GC‐rich wild‐type sequence into a T‐rich sequence could either substantially increase the basal promoter activity and decrease the TPA induction ratio or dramatically reduce the basal promoter activity, depending on the T content. Mutant R5, containing an ATTT sequence at nt − 15 to − 12, caused a more than twofold increase of basal promoter activity and 80% reduction of TPA induction ratio. We suggest that this region interacts with components of the general transcription machinery and that the strength of this interaction is mediated by the T‐content in this region.

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 F1 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.

Involvement of a pertussis-toxin sensitive G protein in the induction of gene expression by insulin☆

Binding of insulin to its receptor triggers multiple cellular responses, including changes in metabolism and in gene expression, resulting from the activation of multiple signalling pathways. Pertussis toxin has been shown to block an insulin-stimulated phospholipase C, resulting in an inhibition of the synthesis of phospholipid second messengers by insulin. In the present study, we investigated the significance of this pathway for the induction of growth-related genes by insulin treatment of H35 hepatoma cells. We found that pertussis toxin dramatically inhibits the induction of c-fos mRNA by insulin. Although c-jun and ornithine decarboxylase induction were also inhibited by pertussis toxin, they were much less sensitive than c-fos. These results indicate an important for lipid second messengers in mitogenic signalling by insulin and further demonstrate distinct roles for this pathway in the induction of c-fos and c-jun.

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.

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.