Jesse D Martinez

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• Centuori, S. M., Gomes, C. J., Trujillo, J., Borg, J., Brownlee, J., Putnam, C. W., & Martinez, J. D. (2016). Deoxycholic acid mediates non-canonical EGFR-MAPK activation through the induction of calcium signaling in colon cancer cells. Biochimica et biophysica acta, 1861(7), 663-70.
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  Obesity and a western diet have been linked to high levels of bile acids and the development of colon cancer. Specifically, increased levels of the bile acid deoxycholic acid (DCA), an established tumor promoter, has been shown to correlate with increased development of colorectal adenomas and progression to carcinoma. Herein we investigate the mechanism by which DCA leads to EGFR-MAPK activation, a candidate mechanism by which DCA may promote colorectal tumorigenesis. DCA treated colon cancer cells exhibited strong and prolonged activation of ERK1/2 when compared to EGF treatment alone. We also showed that DCA treatment prevents EGFR degradation as opposed to the canonical EGFR recycling observed with EGF treatment. Moreover, the combination of DCA and EGF treatment displayed synergistic activity, suggesting DCA activates MAPK signaling in a non-canonical manner. Further evaluation showed that DCA treatment increased intracellular calcium levels and CAMKII phosphorylation, and that blocking calcium with BAPTA-AM abrogated MAPK activation induced by DCA, but not by EGF. Finally we showed that DCA-induced CAMKII leads to MAPK activation through the recruitment of c-Src. Taken together, we demonstrated that DCA regulates MAPK activation through calcium signaling, an alternative mechanism not previously recognized in human colon cancer cells. Importantly, this mechanism allows for EGFR to escape degradation and thus achieve a constitutively active state, which may explain its tumor promoting effects.
• Selmin, O. I., Fang, C., Lyon, A. M., Doetschman, T. C., Thompson, P. A., Martinez, J. D., Smith, J. W., Lance, P. M., & Romagnolo, D. F. (2016). Inactivation of Adenomatous Polyposis Coli Reduces Bile Acid/Farnesoid X Receptor Expression through Fxr gene CpG Methylation in Mouse Colon Tumors and Human Colon Cancer Cells. The Journal of nutrition, 146(2), 236-42.
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  The farnesoid X receptor (FXR) regulates bile acid (BA) metabolism and possesses tumor suppressor functions. FXR expression is reduced in colorectal tumors of subjects carrying inactivated adenomatous polyposis coli (APC). Identifying the mechanisms responsible for this reduction may offer new molecular targets for colon cancer prevention.
• Young, G. M., Radhakrishnan, V. M., Centuori, S. M., Gomes, C. J., & Martinez, J. D. (2015). Comparative analysis of 14-3-3 isoform expression and epigenetic alterations in colorectal cancer. BMC cancer, 15, 826.
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  The 14-3-3 family is a group of intracellular proteins found in all eukaryotic organisms. Humans have seven isoforms that serve as scaffolds to promote interactions of regulatory phospho-proteins involved in many vital cellular processes and previous studies have shown that disturbances in native 14-3-3 levels can contribute significantly to the development of various cancers.
• Centuori, S. M., & Martinez, J. D. (2014). Differential regulation of EGFR-MAPK signaling by deoxycholic acid (DCA) and ursodeoxycholic acid (UDCA) in colon cancer. Digestive diseases and sciences, 59(10), 2367-80.
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  A high-fat diet coincides with increased levels of bile acids. This increase in bile acids, particularly deoxycholic acid (DCA), has been strongly associated with the development of colon cancer. Conversely, ursodeoxycholic acid (UDCA) may have chemopreventive properties. Although structurally similar, DCA and UDCA present different biological and pathological effects in colon cancer progression. The differential regulation of cancer by these two bile acids is not yet fully understood. However, one possible explanation for their diverging effects is their ability to differentially regulate signaling pathways involved in the multistep progression of colon cancer, such as the epidermal growth factor receptor (EGFR)-mitogen-activated protein kinase (MAPK) pathway. This review will examine the biological effects of DCA and UDCA on colon cancer development, as well as the diverging effects of these bile acids on the oncogenic signaling pathways that play a role in colon cancer development, with a particular emphasis on bile acid regulation of the EGFR-MAPK pathway.
• Mount, D. W., Putnam, C. W., Centouri, S. M., Manziello, A. M., Pandey, R., Garland, L. L., & Martinez, J. D. (2014). Using logistic regression to improve the prognostic value of microarray gene expression data sets: application to early-stage squamous cell carcinoma of the lung and triple negative breast carcinoma. BMC medical genomics, 7, 33.
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  Numerous microarray-based prognostic gene expression signatures of primary neoplasms have been published but often with little concurrence between studies, thus limiting their clinical utility. We describe a methodology using logistic regression, which circumvents limitations of conventional Kaplan Meier analysis. We applied this approach to a thrice-analyzed and published squamous cell carcinoma (SQCC) of the lung data set, with the objective of identifying gene expressions predictive of early death versus long survival in early-stage disease. A similar analysis was applied to a data set of triple negative breast carcinoma cases, which present similar clinical challenges.
• Radhakrishnan, V. M., Kojs, P., Young, G., Ramalingam, R., Jagadish, B., Mash, E. A., Martinez, J. D., Ghishan, F. K., & Kiela, P. R. (2014). pTyr421 cortactin is overexpressed in colon cancer and is dephosphorylated by curcumin: involvement of non-receptor type 1 protein tyrosine phosphatase (PTPN1). PloS one, 9(1), e85796.
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  Cortactin (CTTN), first identified as a major substrate of the Src tyrosine kinase, actively participates in branching F-actin assembly and in cell motility and invasion. CTTN gene is amplified and its protein is overexpressed in several types of cancer. The phosphorylated form of cortactin (pTyr(421)) is required for cancer cell motility and invasion. In this study, we demonstrate that a majority of the tested primary colorectal tumor specimens show greatly enhanced expression of pTyr(421)-CTTN, but no change at the mRNA level as compared to healthy subjects, thus suggesting post-translational activation rather than gene amplification in these tumors. Curcumin (diferulolylmethane), a natural compound with promising chemopreventive and chemosensitizing effects, reduced the indirect association of cortactin with the plasma membrane protein fraction in colon adenocarcinoma cells as measured by surface biotinylation, mass spectrometry, and Western blotting. Curcumin significantly decreased the pTyr(421)-CTTN in HCT116 cells and SW480 cells, but was ineffective in HT-29 cells. Curcumin physically interacted with PTPN1 tyrosine phosphatases to increase its activity and lead to dephosphorylation of pTyr(421)-CTTN. PTPN1 inhibition eliminated the effects of curcumin on pTyr(421)-CTTN. Transduction with adenovirally-encoded CTTN increased migration of HCT116, SW480, and HT-29. Curcumin decreased migration of HCT116 and SW480 cells which highly express PTPN1, but not of HT-29 cells with significantly reduced endogenous expression of PTPN1. Curcumin significantly reduced the physical interaction of CTTN and pTyr(421)-CTTN with p120 catenin (CTNND1). Collectively, these data suggest that curcumin is an activator of PTPN1 and can reduce cell motility in colon cancer via dephosphorylation of pTyr(421)-CTTN which could be exploited for novel therapeutic approaches in colon cancer therapy based on tumor pTyr(421)-CTTN expression.
• Martinez, J., Chambers, S. K., & Martinez, J. D. (2012). The significance of p53 isoform expression in serous ovarian cancer. Future oncology (London, England), 8(6).
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  Epithelial ovarian cancer still carries a high mortality rate and remains the leading cause of gynecologic cancer death in the USA, despite decades of research. Hence, there is considerable interest in developing biomarkers that could be used to stratify patients for subsequent treatment. Mutation of the p53 tumor suppressor gene occurs very frequently (∼96%) in high-grade serous ovarian cancer. However, loss of p53 has not proven to be a reliable prognostic marker. Recent evidence indicates that the truncated p53 protein isoforms can regulate activated p53 and thus may play a role in tumorigenesis. In the article by Hofstetter et al., the authors examined the relationship between the expression of two p53 isoforms (Δ133p53 and Δ40p53) and prognosis in patients with serous ovarian cancer. Their findings indicate that Δ133p53 constitutes an independent prognostic marker for improved recurrence-free and overall survival. Intriguingly, this relationship was observed in patients whose tumors expressed a mutant p53, suggesting that Δ133p53 might suppress the actions of mutant p53. The mutational status of p53 alone did not have prognostic significance. These studies suggest that mutant p53 activity may be counteracted by Δ133p53, which leads to a more favorable prognosis in advanced serous ovarian carcinomas. Novel therapeutic approaches could be built upon these findings.
• Martinez, J., Radhakrishnan, V. M., Putnam, C. W., & Martinez, J. D. (2012). Activation of phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signaling and the consequent induction of transformation by overexpressed 14-3-3γ protein require specific amino acids within 14-3-3γ N-terminal variable region II. The Journal of biological chemistry, 287(52).
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  Members of the 14-3-3 superfamily regulate numerous cellular functions by binding phosphoproteins. The seven human isoforms (and the myriad of other eukaryotic 14-3-3 proteins) are highly conserved in amino acid sequence and secondary structure, yet there is abundant evidence that the various isoforms manifest disparate as well as common functions. Several of the human 14-3-3 isoforms are dysregulated in certain cancers and thus have been implicated in oncogenesis; experimentally, 14-3-3γ behaves as an oncogene, whereas 14-3-3σ acts as a tumor suppressor. In this study, we sought to localize these opposing phenotypes to specific regions of the two isoforms and then to individual amino acids therein. Using a bioinformatics approach, six variable regions (VRI-VRVI) were identified. Using this information, two sets of constructs were created in which N-terminal portions (including either VRI-IV or only VRI and VRII) of 14-3-3γ and 14-3-3σ were swapped; NIH3T3 cells overexpressing the four chimeric proteins were tested for transformation activity (focus formation, growth in soft agar) and activation of PI3K and MAPK signaling. We found that the specific phenotypes of 14-3-3γ are associated with the N-terminal 40 amino acids (VRI and VRII); in like fashion, VRI and VRII of 14-3-3σ dictated its tumor suppressor function. Using individual amino acid substitutions within the 14-3-3γ VRII, we identified two residues required for and two contributing to the γ-specific phenotypes. Our observations suggest that isoform-specific phenotypes are dictated by a relatively few amino acids within variable regions.
• Martinez, J., Li, Q., & Martinez, J. D. (2011). Loss of HSF1 results in defective radiation-induced G(2) arrest and DNA repair. Radiation research, 176(1).
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  HSF1 is a transcription factor that plays a key role in the response to heat stress and was previously shown by us to also be essential for importation of p53 into the nucleus. Here we extend these studies and show that loss of HSF1 renders cells more sensitive to killing by ionizing radiation. Cells that lack a functional HSF1 were unable to arrest in G(2) after exposure to ionizing radiation, suggesting that HSF1 activity was essential for activation of this cell cycle checkpoint. In addition, cells with no HSF1 showed a reduced capacity to repair radiation-induced double-stranded DNA breaks. We found that in these cells 53BP1 did not accumulate at sites of DNA damage, suggesting that HSF1 was also essential for the functioning of this DNA damage mediator. Taken together our results indicate that HSF1 plays an important role in checkpoint activation and DNA repair and suggest that there is overlap between the heat stress response pathway and the pathway that responds to ionizing radiation.
• Martinez, J., Li, Q., & Martinez, J. D. (2011). P53 is transported into the nucleus via an Hsf1-dependent nuclear localization mechanism. Molecular carcinogenesis, 50(2).
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  Loss of p53 function can occur through disruption of its ability to localize to the nucleus. Previously we showed through characterization a set of mutant cell lines that lacked the ability to import p53 into the nucleus that nuclear translocation of p53 appeared to be mechanistically different from that of the SV40 T-antigen (SV40TAg). Here we extend that work by examining nuclear importation of p53 and SV40TAg using both in vivo and in vitro assays for nuclear localization. We show that disruption of microtubule polymerization using colchicine suppresses nuclear localization of p53 but not of SV40TAg. We also show, for the first time, that the heat shock transcription factor (Hsf1), is required for establishment of the microtubule network in cells and for nuclear localization of p53. In contrast, SV40TAg does not interact with polymerized microtubules suggesting that it is transported into the nucleus through an alternative mechanism. Interestingly, lacking of Hsf1 expression and suppressing Hsf1 by siRNA also made cells more resistant to the cytotoxic effects of paclitaxel. Hence, loss of Hsf1 activity not only suppressed p53 function, but also led to reduced sensitivity to killing by drugs that target microtubules.
• Martinez, J., Radhakrishnan, V. M., Putnam, C. W., Qi, W., & Martinez, J. D. (2011). P53 suppresses expression of the 14-3-3 gamma oncogene. BMC cancer, 11.
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  14-3-3 proteins are a family of highly conserved proteins that are involved in a wide range of cellular processes. Recent evidence indicates that some of these proteins have oncogenic activity and that they may promote tumorigenesis. We previously showed that one of the 14-3-3 family members, 14-3-3 gamma, is over expressed in human lung cancers and that it can induce transformation of rodent cells in vitro.
• Martinez, J., & Martinez, J. D. (2010). Restoring p53 tumor suppressor activity as an anticancer therapeutic strategy. Future oncology (London, England), 6(12).
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  Loss of p53 tumor suppressor function is a key event in the genesis of most human tumors. This observation has prompted efforts to restore p53 activity as an anticancer therapeutic approach. Recent developments that have extended our understanding of how p53 activity is regulated and how mutations disrupt that regulation have provided the insight needed to develop therapeutic strategies that take advantage of this knowledge. In this article, we review the strategies for restoring p53 function and some of the new compounds that show promise as antitumor agents in preclinical models.
• Martinez, J., Radhakrishnan, V. M., & Martinez, J. D. (2010). 14-3-3gamma induces oncogenic transformation by stimulating MAP kinase and PI3K signaling. PloS one, 5(7).
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  The 14-3-3 proteins are a set of highly conserved scaffolding proteins that have been implicated in the regulation of a variety of important cellular processes such as the cell cycle, apoptosis and mitogenic signaling. Recent evidence indicates that the expression of some of the family members is elevated in human cancers suggesting that they may play a role in tumorigenesis. In the present study, the oncogenic potential of 14-3-3gamma was shown by focus formation and tumor formation in SCID mice using 14-3-3gamma transfected NIH3T3 mouse fibroblast cells. In contrast, 14-3-3sigma, a putative tumor suppressor, inhibited NIH3T3 transformation by H-ras and c-myc. We also report that activation of both MAP kinase and PI3K signaling pathways are essential for transformation by 14-3-3gamma. In addition, we found that 14-3-3gamma interacts with phosphatidylinositol 3-kinase (PI3K) and TSC2 proteins indicating that it could stimulate PI3K signaling by acting at two points in the signaling pathway. Overall, our studies establish 14-3-3gamma as an oncogene and implicate MAPK and PI3K signaling as important for 14-3-3gamma induced transformation.
• Martinez, J., Feldman, R., & Martinez, J. D. (2009). Growth suppression by ursodeoxycholic acid involves caveolin-1 enhanced degradation of EGFR. Biochimica et biophysica acta, 1793(8).
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  Ursodeoxycholic acid (UDCA) has been shown to prevent colon tumorigenesis in animal models and in humans. In vitro work indicates that this bile acid can suppress cell growth and mitogenic signaling suggesting that UDCA may be an anti-proliferative agent. However, the mechanism by which UDCA functions is unclear. Previously we showed that bile acids may alter cellular signaling by acting at the plasma membrane. Here we utilized EGFR as a model membrane receptor and examined the effects that UDCA has on its functioning. We found that UDCA promoted an interaction between EGFR and caveolin-1 and this interaction enhanced UDCA-mediated suppression of MAP kinase activity and cell growth. Importantly, UDCA treatment led to recruitment of the ubiquitin ligase, c-Cbl, to the membrane, ubiquitination of EGFR, and increased receptor degradation. Moreover, suppression of c-Cbl activity abrogated UDCA's growth suppression activities suggesting that receptor ubiquitination plays an important role in UDCA's biological activities. Taken together these results suggest that UDCA may act to suppress cell growth by inhibiting the mitogenic activity of receptor tyrosine kinases such as EGFR through increased receptor degradation.
• Junk, D. J., Vrba, L., Watts, G. S., Oshiro, M. M., Martinez, J. D., & Futscher, B. W. (2008). Different mutant/wild-type p53 combinations cause a spectrum of increased invasive potential in nonmalignant immortalized human mammary epithelial cells. Neoplasia (New York, N.Y.), 10(5), 450-61.
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  Aberrations of p53 occur in most, if not all, human cancers. In breast cancer, p53 mutation is the most common genetic defect related to a single gene. Immortalized human mammary epithelial cells resemble the earliest forms of aberrant breast tissue growth but do not express many malignancy-associated phenotypes. We created a model of human mammary epithelial tumorigenesis by infecting hTERT-HME1 immortalized human mammary epithelial cells expressing wild-type p53 with four different mutant p53 constructs to determine the role of p53 mutation on the evolution of tumor phenotypes. We demonstrate that different mutant/wild-type p53 heterozygous models generate loss of function, dominant negative activity, and a spectrum of gain of function activities that induce varying degrees of invasive potential. We suggest that this model can be used to elucidate changes that occur in early stages of human mammary epithelial tumorigenesis. These changes may constitute novel biomarkers or reveal novel treatment modalities that could inhibit progression from primary to metastatic breast disease.
• Martinez, J., Hong, S. W., Qi, W., Brabant, M., Bosco, G., & Martinez, J. D. (2008). Human 14-3-3 gamma protein results in abnormal cell proliferation in the developing eye of Drosophila melanogaster. Cell division, 3.
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  14-3-3 proteins are a family of adaptor proteins that participate in a wide variety of cellular processes. Recent evidence indicates that the expression levels of these proteins are elevated in some human tumors providing circumstantial evidence for their involvement in human cancers. However, the mechanism through which these proteins act in tumorigenesis is uncertain.
• Martinez, J., Li, Q., Feldman, R. A., Radhakrishnan, V. M., Carey, S., & Martinez, J. D. (2008). Hsf1 is required for the nuclear translocation of p53 tumor suppressor. Neoplasia (New York, N.Y.), 10(10).
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  Although the p53 tumor suppressor is most frequently inactivated by genetic mutations, exclusion from the nucleus is also seen in human tumors. We have begun to examine p53 nuclear importation by isolating a series of mutant cells in which the temperature-sensitive murine p53(Val135) mutant is sequestered in the cytoplasm. We previously showed that that three of them (ALTR12, ALTR19, and ALTR25) constituted a single complementation group. Here, we found that ALTR12 cells are more sensitive to heat stress than either ALTR19 or ALTR25 and that there was a complete lack of induction of Hsp70 in response to heat shock. Western blot analysis showed no expression of the Hsf1 transcription factor, and neither heat shock nor azetidine could induce p53 nuclear localization in ALTR12 cells but did in parental A1-5 cells. Suppression of Hsf1 in A1-5 cells with quercetin or an Hsf1 siRNA reduced p53 nuclear importation and inhibited p53-mediated activation of a p21 reporter. Most convincingly, p53 nuclear importation could be restored in ALTR12 cells by introducing an exogenous Hsf1 gene. Collectively, our result suggests that Hsf1 is required for p53 nuclear importation and activation and implies that heat shock factors play a role in the regulation of p53.
• Li, Q., Falsey, R. R., Gaitonde, S., Sotello, V., Kislin, K., & Martinez, J. D. (2007). Genetic analysis of p53 nuclear importation. Oncogene, 26(57), 7885-93.
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  A key step in activation of the p53 tumor suppressor is its transport into the nucleus; however, despite intensive study of p53, the regulation of its subcellular localization is still poorly understood. Here we examined the p53 nuclear importation using a series of mutant cell lines that were resistant to the growth inhibitory effects of temperature-sensitive murine p53 (tsp53). Examination of the p53 subcellular localization in these cell lines showed that the protein was cytoplasmic in most of them. Using a digitonin-permeabilized cell in vitro nuclear import system, we show that cytosols from these cell lines do not support nuclear translocation of a p53 nuclear localization signal (NLS)-containing substrate protein, but promote nuclear localization of a SV40TAgNLS-containing substrate. Complementation assays and use of the mutant cells themselves in the in vitro assays demonstrate that both soluble and insoluble protein components are involved in p53 nuclear import. Collectively, our results suggest that there is a p53 NLS-selective nuclear import pathway and that both soluble and insoluble proteins are involved in its function.
• Martinez, J., Li, X., Chen, H., Huang, Z., Su, H., & Martinez, J. D. (2007). Global mapping of gene/protein interactions in PubMed abstracts: a framework and an experiment with P53 interactions. Journal of biomedical informatics, 40(5).
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  Gene/protein interactions provide critical information for a thorough understanding of cellular processes. Recently, considerable interest and effort has been focused on the construction and analysis of genome-wide gene networks. The large body of biomedical literature is an important source of gene/protein interaction information. Recent advances in text mining tools have made it possible to automatically extract such documented interactions from free-text literature. In this paper, we propose a comprehensive framework for constructing and analyzing large-scale gene functional networks based on the gene/protein interactions extracted from biomedical literature repositories using text mining tools. Our proposed framework consists of analyses of the network topology, network topology-gene function relationship, and temporal network evolution to distill valuable information embedded in the gene functional interactions in the literature. We demonstrate the application of the proposed framework using a testbed of P53-related PubMed abstracts, which shows that the literature-based P53 networks exhibit small-world and scale-free properties. We also found that high degree genes in the literature-based networks have a high probability of appearing in the manually curated database and genes in the same pathway tend to form local clusters in our literature-based networks. Temporal analysis showed that genes interacting with many other genes tend to be involved in a large number of newly discovered interactions.
• Martinez, J., Meade-Tollin, L., & Martinez, J. D. (2007). Loss of p53 and overexpression of EphA2 predict poor prognosis for ovarian cancer patients. Cancer biology & therapy, 6(2).
• Martinez, J., Qi, W., Liu, X., Chen, W., Li, Q., & Martinez, J. D. (2007). Overexpression of 14-3-3gamma causes polyploidization in H322 lung cancer cells. Molecular carcinogenesis, 46(10).
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  The 14-3-3 proteins are a family of highly conserved proteins that participate in a wide variety of cellular processes. Mounting evidence suggests that 14-3-3 proteins have a role in human cancers, however their role in tumorigenesis is unclear. Here we report that over-expression of 14-3-3 gamma protein in human lung cancer cell line H322 results in abnormal DNA replication and polyploidization. Cells that overexpress 14-3-3 gamma are resistant to microtubule inhibitors and can reenter the cell cycle in the absence of mitosis suggesting that elevated levels of 14-3-3 gamma may enable cells to bypass the mitotic checkpoint. Taken together, our data indicate that 14-3-3gamma may contribute to tumorigenesis by promoting genomic instability.
• Martinez, J., Akare, S., Jean-Louis, S., Chen, W., Wood, D. J., Powell, A. A., & Martinez, J. D. (2006). Ursodeoxycholic acid modulates histone acetylation and induces differentiation and senescence. International journal of cancer. Journal international du cancer, 119(12).
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  Agents that can modulate colonic environment and control dysregulated signaling are being evaluated for their chemopreventive potential in colon cancer. Ursodeoxycholate (UDCA) has shown chemopreventive potential in preclinical and animal models of colon cancer, but the mechanism behind it remains unknown. Here biological effects of UDCA were examined to understand mechanism behind its chemoprevention in colon cancer. Our data suggests that UDCA can suppress growth in a wide variety of cancer cell lines and can induce low level of apoptosis in colon cancer cells. We also found that UDCA treatment induces alteration in morphology, increased cell size, upregulation of cytokeratin 8, 18 and 19 and E-cadherin, cytokeratin remodeling and accumulation of lipid droplets, suggesting that UDCA induces differentiation in colon carcinoma cells. Our results also suggest significant differences in UDCA and sodium butyrate induced functional differentiation. We also report for the first time that UDCA can induce senescence in colon cancer cells as assessed by flattened, spread out and vacuolated morphology as well as by senescence marker beta-galactosidase staining. We also found that UDCA inhibits the telomerase activity. Surprisingly, we found that UDCA is not a histone deacytylase inhibitor but instead induces hypoacetylation of histones unlike hyperacetylation induced by sodium butyrate. Our results also suggest that, although UDCA induced senescence is p53, p21 and Rb independent, HDAC6 appears to be important in UDCA induced senescence. In summary, our data shows that UDCA modulates chromatin by inducing histone hypoacetylation and induces differentiation and senescence in colon cancer cells.
• Martinez, J., Jean-Louis, S., Akare, S., Ali, M. A., Mash, E. A., Meuillet, E., & Martinez, J. D. (2006). Deoxycholic acid induces intracellular signaling through membrane perturbations. The Journal of biological chemistry, 281(21).
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  Secondary bile acids have long been postulated to be tumor promoters in the colon; however, their mechanism of action remains unclear. In this study, we examined the actions of bile acids at the cell membrane and found that they can perturb membrane structure by alteration of membrane microdomains. Depletion of membrane cholesterol by treating with methyl-beta-cyclodextrin suppressed deoxycholic acid (DCA)-induced apoptosis, and staining for cholesterol with filipin showed that DCA caused a marked rearrangement of this lipid in the membrane. Likewise, DCA was found to affect membrane distribution of caveolin-1, a marker protein that is enriched in caveolae membrane microdomains. Additionally, fluorescence anisotropy revealed that DCA causes a decrease in membrane fluidity consistent with the increase in membrane cholesterol content observed after 4 h of DCA treatment of HCT116 cells. Significantly, by using radiolabeled bile acids, we found that bile acids are able to interact with and localize to microdomains differently depending on their physicochemical properties. DCA was also found to induce tyrosine phosphorylation and activate the receptor tyrosine kinase epidermal growth factor receptor in a ligand-independent manner. In contrast, ursodeoxycholic acid did not exhibit any of these effects even though it interacted significantly with the microdomains. Collectively, these data suggest that bile acid-induced signaling is initiated through alterations of the plasma membrane structure and the redistribution of cholesterol.
• Martinez, J., Powell, A. A., Akare, S., Qi, W., Herzer, P., Jean-Louis, S., Feldman, R. A., & Martinez, J. D. (2006). Resistance to ursodeoxycholic acid-induced growth arrest can also result in resistance to deoxycholic acid-induced apoptosis and increased tumorgenicity. BMC cancer, 6.
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  There is a large body of evidence which suggests that bile acids increase the risk of colon cancer and act as tumor promoters, however, the mechanism(s) of bile acids mediated tumorigenesis is not clear. Previously we showed that deoxycholic acid (DCA), a tumorogenic bile acid, and ursodeoxycholic acid (UDCA), a putative chemopreventive agent, exhibited distinct biological effects, yet appeared to act on some of the same signaling molecules. The present study was carried out to determine whether there is overlap in signaling pathways activated by tumorogenic bile acid DCA and chemopreventive bile acid UDCA.
• Martinez, J., & Martinez, J. D. (2005). 14-3-3 proteins: do they have a role in human cancer?. Future oncology (London, England), 1(5).
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  The 14-3-3 proteins are a family of adaptors that have recently gained a lot of attention due to the discovery that they may play an important role in human tumorigenesis. Examination of human tumors shows that expression of these proteins is increased in some tumors and decreased in others. Since biochemical studies suggest that 14-3-3 proteins regulate multiple pathways that are also disrupted during tumorigenesis, it is unclear how they actually contribute to tumor development.
• Martinez, J., Akare, S., & Martinez, J. D. (2005). Bile acid induces hydrophobicity-dependent membrane alterations. Biochimica et biophysica acta, 1735(1).
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  Elevated concentrations of fecal bile acids are a known risk factor for colon cancer, owing to alterations in cellular signaling. In colonic cells, where bile acid uptake is minimal, the hydrophobicity-induced membrane perturbation and alterations have been proposed, but these membrane alterations are largely uncharacterized. In this study, we examined the determinants and characteristics of bile acid-induced membrane alterations, utilizing PKCalpha activation and cholesterol up-regulation as model indicators. We found that bile acid-induced PKCalpha activation is a function of hydrophobicity and correlated with alteration in membrane lipid composition, as evident by the significant up-regulation in membrane cholesterol and phospholipid. We found that bile acid do not cause cell membrane disruption at a concentration sufficient to activate PKCalpha, but do induce drastic alterations in membrane composition. Bile acid also induced the modification and up-regulation of caveolin-1 in a hydrophobicity-dependent manner, implying widespread receptor dysregulation. Similarly, ERK1/2 activation was observed only in response to hydrophobic bile acids, suggesting hydrophobicity-induced caveolar or membrane stress. Experiments with sodium lauryl sarcosine and cholesteryl hemisuccinate showed that bile acid-induced membrane alterations can be mimicked by hydrophobic molecules unrelated to bile acids, strongly implicating hydrophobicity as an important determinant of bile acid signaling.
• Martinez, J., Im, E., Akare, S., Powell, A., & Martinez, J. D. (2005). Ursodeoxycholic acid can suppress deoxycholic acid-induced apoptosis by stimulating Akt/PKB-dependent survival signaling. Nutrition and cancer, 51(1).
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  The nontoxic bile acid ursodeoxycholic acid (UDCA) is reported to be an anti-apoptotic agent with efficacy against a variety of death stimuli including the cytotoxic bile acid deoxycholic acid (DCA). To gain insight into this anti-apoptotic property, we tested UDCA for its ability to protect the colon carcinoma-derived cell line HCT116 against DCA-induced apoptosis. We found that UDCA could suppress DCA-induced apoptosis in a time- and dose-dependent manner and that this effect correlated with Akt phosphorylation. Importantly, UDCA lost its ability to protect cells from DCA-induced cell death when Akt activity was suppressed genetically using a dominant negative Akt mutant or when PI3K activity was inhibited pharmacologically. These results suggest that UDCA can protect HCT116 cells against DCA-induced apoptosis by stimulating Akt-dependent survival signaling.
• Martinez, J., Qi, W., Liu, X., Qiao, D., & Martinez, J. D. (2005). Isoform-specific expression of 14-3-3 proteins in human lung cancer tissues. International journal of cancer. Journal international du cancer, 113(3).
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  14-3-3 Proteins play important roles in a wide range of vital regulatory processes, including signal transduction, apoptosis, cell cycle progression and DNA replication. In mammalian cells, 7 14-3-3 isoforms (beta, gamma, epsilon, eta, sigma, theta and zeta) have been identified and each of these seems to have distinct tissue localizations and isoform-specific functions. Previous studies have shown that 14-3-3 protein levels are higher in human lung cancers as compared to normal tissues. It is unclear, however, which of the 14-3-3 isoform(s) are overexpressed in these cancers. In our study, the levels of all seven 14-3-3 isoforms were examined by RT-PCR and Western blotting. We show that the message for only two isoforms, 14-3-3epsilon and zeta, could be detected in normal tissues. In lung cancer biopsies, however, four isoforms, 14-3-3beta, gamma, sigma, and theta;, in addition to 14-3-3epsilon and zeta, were present in abundance. The expression frequency of 14-3-3beta, gamma, sigma and theta; isoforms was 11, 10, 13 and 8 of the 14 biopsies examined, respectively. The data from immunohistochemical staining and Western blotting were consistent with the RT-PCR results. Given the prevalence of elevated 14-3-3 expression in human lung cancers we propose that these proteins may be involved in lung cancer tumorigenesis and that specific 14-3-3 proteins may be useful as markers for lung cancer diagnosis and targets for therapy.
• Martinez, J., Im, E., & Martinez, J. D. (2004). Ursodeoxycholic acid (UDCA) can inhibit deoxycholic acid (DCA)-induced apoptosis via modulation of EGFR/Raf-1/ERK signaling in human colon cancer cells. The Journal of nutrition, 134(2).
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  Ursodeoxycholic acid (UDCA), a hydrophilic bile acid, is known as a cytoprotective agent. UDCA prevents apoptosis induced by a variety of stress stimuli including cytotoxic bile acids such as deoxycholic acid (DCA). Here we examined the molecular mechanism by which UDCA can antagonize DCA-induced apoptosis in human colon cancer cells. UDCA pretreatment decreases the number of apoptotic cells caused by exposure to DCA and UDCA. Further studies of the signaling pathway showed that UDCA pretreatment suppressed DNA binding activity of activator protein-1 and this was accompanied by downregulation of both extracellular signal-regulated kinase (ERK) and Raf-1 kinase activities stimulated by exposure to DCA. DCA was also found to activate epidermal growth factor receptor (EGFR) activity and UDCA inhibited this. Collectively, these findings suggest that the inhibitory effect of UDCA in DCA-induced apoptosis is partly mediated by modulation of EGFR/Raf-1/ERK signaling.
• Martinez, J., Leroy, G., Chen, H., & Martinez, J. D. (2003). A shallow parser based on closed-class words to capture relations in biomedical text. Journal of biomedical informatics, 36(3).
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  Natural language processing for biomedical text currently focuses mostly on entity and relation extraction. These entities and relations are usually pre-specified entities, e.g., proteins, and pre-specified relations, e.g., inhibit relations. A shallow parser that captures the relations between noun phrases automatically from free text has been developed and evaluated. It uses heuristics and a noun phraser to capture entities of interest in the text. Cascaded finite state automata structure the relations between individual entities. The automata are based on closed-class English words and model generic relations not limited to specific words. The parser also recognizes coordinating conjunctions and captures negation in text, a feature usually ignored by others. Three cancer researchers evaluated 330 relations extracted from 26 abstracts of interest to them. There were 296 relations correctly extracted from the abstracts resulting in 90% precision of the relations and an average of 11 correct relations per abstract.
• Martinez, J., Qi, W., & Martinez, J. D. (2003). Reduction of 14-3-3 proteins correlates with increased sensitivity to killing of human lung cancer cells by ionizing radiation. Radiation research, 160(2).
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  The 14-3-3 proteins have a wide range of ligands and are involved in a variety of biological pathways. Importantly, 14-3-3 proteins are known to be overexpressed in some human lung cancers, suggesting that they may play a role in tumorigenesis. Here we examined 14-3-3 expression in several lung cancer-derived cell lines and found that four of the seven 14-3-3 isoforms, beta, epsilon, theta and zeta, were highly expressed in both lung cancer cell lines and normal lung fibroblasts. Two isoforms, sigma and gamma, were present only at very low levels. Immunoprecipitation data showed 14-3-3zeta could bind to CDC25C in irradiated A549 cells, and suppression of 14-3-3zeta in A549 cells with antisense resulted in a decrease in CDC25C localization in cytoplasm and CDC2 phosphorylation on Tyr15. As a consequence, CDC2 activity remained elevated which resulted in release from radiation-induced G(2)/M-phase arrest. Moreover, 16% 14-3-3zeta antisense-transfected cells underwent apoptosis when exposed to 10 Gy ionizing radiation. These data indicate that 14-3-3zeta is involved in G(2) checkpoint activation and that inhibition of 14-3-3 may be a useful approach to sensitize human lung cancers to ionizing radiation.
• Martinez, J., Stea, B., Falsey, R., Kislin, K., Patel, J., Glanzberg, H., Carey, S., Ambrad, A. A., Meuillet, E. J., & Martinez, J. D. (2003). Time and dose-dependent radiosensitization of the glioblastoma multiforme U251 cells by the EGF receptor tyrosine kinase inhibitor ZD1839 ('Iressa'). Cancer letters, 202(1).
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  Hyperactive epidermal growth factor receptor (EGFR) signaling, which promotes unregulated cell growth and inhibits apoptosis, is believed to contribute to clinical radiation resistance of glioblastoma multiforme (GBM). Blockage of the EGFR signalling pathways may offer an attractive therapeutic target to increase the cytotoxic effects of radiotherapy. We report the effects of ZD1839 ('Iressa'), a selective EGFR tyrosine kinase inhibitor on the radiation sensitivity of the U251 GBM cell line, which expresses high levels of EGFR. In radiation survival experiments, 5 microM of ZD1839 had a significant radiosensitizing effect and increased cell death was observed at doses of 5Gy in the presence of ZD1839. Dose and schedule of drug administration in combination with radiation appeared to be a crucial element to obtain radiosensitization of the cells. These studies suggest novel therapeutic strategies in the treatment of GBM.
• Martinez, J., Qi, W., Qiao, D., & Martinez, J. D. (2002). Caffeine induces TP53-independent G(1)-phase arrest and apoptosis in human lung tumor cells in a dose-dependent manner. Radiation research, 157(2).
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  Caffeine is a model radiosensitizing agent that is thought to work by abrogating the radiation-induced G(2)-phase checkpoint. In this study, we examined the effect that various concentrations of caffeine had on cell cycle checkpoints and apoptosis in cells of a human lung carcinoma cell line and found that a concentration of 0.5 mM caffeine could abrogate the G(2)-phase arrest normally seen after exposure to ionizing radiation. Surprisingly, at a concentration of 5 mM, caffeine not only induced apoptosis by itself and acted synergistically to enhance radiation-induced apoptosis, but also induced a TP53-independent G(1)-phase arrest. Examination of the molecular mechanisms by which caffeine produced these effects revealed that caffeine had opposing effects on different cyclin-dependent kinases. CDK2 activity was suppressed by caffeine, whereas activity of CDC2 was enhanced by suppressing phosphorylation on Tyr15 and by interfering with 14-3-3 binding to CDC25C. These data indicate that the effect of caffeine on cell cycle checkpoints and apoptosis is dependent on dose and that caffeine acts through differential regulation of cyclin-dependent kinase activity.
• Martinez, J., Martinez, J. D., McNeill, K. M., Capp, M. P., Dallas, W. J., & Martinez, J. D. (1992). Standardization of network addressing in picture archiving and communications systems utilizing the ISO OSI protocols. Computer methods and programs in biomedicine, 37(4).
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  The establishment of a communications network requires the definition of addressing schemes to identify systems attached to the network. When it is anticipated that such a network will interconnect to, or communicate with, other similar networks it is beneficial to define standardized addressing schemes. This paper discusses the need for the standardization of network service access point (NSAP) addresses in picture archiving and communications systems (PACS) which use the open systems interconnect (OSI) communications protocols. Possible methods for establishing the necessary hierarchy of address registration authorities are discussed, along with the corresponding address formats.

Poster Presentations

• Gilman, K. A., Charles, P. W., Mount, D. W., & Martinez, J. D. (2016, April). Modeling combinatorial chemotherapy in vitro using Saccharomyces cerevisiae and human cell lines. University of Arizona Cancer Center Retreat. Tucson, Arizona: University of Arizona Cancer Center.
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  Modeling Combinatorial Chemotherapy in Vitro Using Saccharomyces cerevisiae and Human Cell LinesKaitlyn A. Gilman, Department of Surgery, The University of Arizona (Presenter)Charles W. Putnam, Department of Surgery, The University of Arizona (Co-Investigator)David W. Mount, Department of Molecular and Cellular Biology Jesse D. Martinez, Department of Cellular and Molecular Medicine, The University of Arizona (Principal Investigator)BackgroundStatins, the most commonly prescribed medications worldwide, have shown variable anti-tumor effects in cancers of the breast, colon, lung, liver, pancreas, bladder, ovary, and prostate. However, attempts to demonstrate synergistic effects when combined with chemotherapeutic drugs have yielded inconsistent results. In this study we used Saccharomyces cerevisiae, a well-established model for anticancer drug research, to better characterize the pharmacologic interactions between lovastatin and ten drugs commonly used in chemotherapy. Additionally, we tested lovastatin plus cisplatin in four human cancer cell lines. MethodsWe evaluated lovastatin in two-drug combinations with 5-FU, cisplatin, cyclophosphamide, doxorubicin, etoposide, epothilone, gemcitabine, methotrexate, rapamycin, and tamoxifen. A pool of Saccharomyces cerevisiae diploid strains heterozygously deleted for each essential gene was compiled. Using 96 well plates, we performed serial 70% dilutions in a 7 X 7 matrix of rows (lovastatin) and columns (Drug B). After adding pooled yeast cells to each well, the plates were incubated with shaking at 30° C. Optical densities were recorded with a plate reader at 36 and 48 h. Three to seven replicates were performed for all drug combinations. The data were analyzed according to the Loewe Model using Combenefit© Software (Version 2.021; Sourceforge, 2014). To evaluate the effects of lovastatin and cisplatin in human cell lines, assays were performed in a similar format with MCF7 (breast cancer), HT29 (colon cancer), HepG2 (hepatocellular carcinoma) and A549 (lung adenocarcinoma) cells. ResultsIn yeast, the effects of lovastatin were quite variable, depending upon (1) the chemotherapeutic drug partner, (2) the concentrations of the two drugs; and (3), to a lesser degree, the time point. Surprisingly, when paired with some drugs (e.g., methotrexate), lovastatin displayed both antagonism and synergism. Interestingly, global antagonism (e.g., gemcitabine) and strong synergism (e.g., rapamycin and tamoxifen) were also observed. With other drugs, lovastatin was generally additive but with synergism or antagonism evident in narrow ranges of concentration. In human cell culture, the interactions of lovastatin and cisplatin varied with cell type but were consistently antagonistic at lower concentrations of lovastatin. Lovastatin and cisplatin were strongly antagonistic in MCF7 and A549 cells. However, when tested in HT29 cells, lovastatin and cisplatin showed a dual effect of very strong antagonism at low concentrations and weak synergism at higher lovastatin concentrations. Conclusions and Translational SignificanceAlthough lovastatin shows promise in augmenting the effects of anti-cancer drugs, our data illuminate inconsistencies regarding the reported effects of statins. That lower concentrations of lovastatin were antagonistic with cisplatin in all human cancer lines tested argues against the indiscriminate clinical use of lovastatin in combination with chemotherapeutic drugs without additional experimental support. This project will be expanded to further evaluate the effects of lovastatin with rapamycin and tamoxifen, to test lovastatin in three-drug combinations, and to identify the yeast mutations conferring resistance to various drug combinations.