George S Watts
- Associate Research Scientist, Cancer Center Division
- Ph.D. Pharmacology/Toxicology
- University of Arizona, Tucson, Arizona, United States
- B.S. Chemistry
- University of Delaware, Newark, Delaware, United States
- Associate Professor, University of Arizona, Tucson, Arizona (2000 - Ongoing)
Diagnosis of infectious pathogens by whole genome sequencing.
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- Watts, G. S., Youens-Clark, K., Slepian, M. J., Wolk, D. M., Oshiro, M. M., Metzger, G. S., Dhingra, D., Cranmer, L. D., & Hurwitz, B. L. (2017). 16S rRNA gene sequencing on a benchtop sequencer: accuracy for identification of clinically important bacteria. Journal of applied microbiology, 123(6), 1584-1596.More infoTest the choice of 16S rRNA gene amplicon and data analysis method on the accuracy of identification of clinically important bacteria utilizing a benchtop sequencer.
- Harshman, D. K., Rao, B. M., McLain, J. E., Watts, G. S., & Yoon, J. Y. (2015). Innovative qPCR using interfacial effects to enable low threshold cycle detection and inhibition relief. Science advances, 1(8), e1400061.More infoMolecular diagnostics offers quick access to information but fails to operate at a speed required for clinical decision-making. Our novel methodology, droplet-on-thermocouple silhouette real-time polymerase chain reaction (DOTS qPCR), uses interfacial effects for droplet actuation, inhibition relief, and amplification sensing. DOTS qPCR has sample-to-answer times as short as 3 min 30 s. In infective endocarditis diagnosis, DOTS qPCR demonstrates reproducibility, differentiation of antibiotic susceptibility, subpicogram limit of detection, and thermocycling speeds of up to 28 s/cycle in the presence of tissue contaminants. Langmuir and Gibbs adsorption isotherms are used to describe the decreasing interfacial tension upon amplification. Moreover, a log-linear relationship with low threshold cycles is presented for real-time quantification by imaging the droplet-on-thermocouple silhouette with a smartphone. DOTS qPCR resolves several limitations of commercially available real-time PCR systems, which rely on fluorescence detection, have substantially higher threshold cycles, and require expensive optical components and extensive sample preparation. Due to the advantages of low threshold cycle detection, we anticipate extending this technology to biological research applications such as single cell, single nucleus, and single DNA molecule analyses. Our work is the first demonstrated use of interfacial effects for sensing reaction progress, and it will enable point-of-care molecular diagnosis of infections.
- Jandova, J., Mason, C. J., Pawar, S. C., & Watts, G. S. (2015). Fn14 receptor promotes invasive potential and metastatic capacity of non-small lung adenocarcinoma cells through the up-regulation of integrin α6. Neoplasma, 62(1), 41-52.More infoLung cancer is one of the leading cause of cancer-related death around the world with the majority of diagnoses being non-small cell lung cancer (NSCLC). Given the poor survival rate and efficacy of current therapy for NSCLC, there is a need to identify and develop new therapeutic targets for treatment. We have observed significantly up-regulated levels of Fn14 in clinical samples of lung cancer relative to normal adjacent tissue. However, the functional role of Fn14 in these tumors is not understood yet. We used RT-PCR to establish the Fn14 expression profile in various NSCLC cell lines. Using isogenic variants of H460 NSCLC cell line with low, intermediate and high Fn14 expression as a cellular model, we determined that increased levels of integrin α6 in cells over-expressing Fn14 is suggestive of an important role of α6β1-fn14 interactions in motility of lung carcinoma and formation of metastases. Enhanced levels of Fn14 correlated with higher tumor cell migration and invasion in an MMP-1 dependent manner. Cells over-expressing Fn14 showed increased in vivo tumor formation with metastatic capacity to lymph nodes, lungs and liver. Thus, this research may be a step toward developing improved treatment strategies for NSCLC by improved detection and inhibition of metastases.
- Garbe, J. C., Vrba, L., Sputova, K., Fuchs, L., Novak, P., Brothman, A. R., Jackson, M., Chin, K., LaBarge, M. A., Watts, G., Futscher, B. W., & Stampfer, M. R. (2014). Immortalization of normal human mammary epithelial cells in two steps by direct targeting of senescence barriers does not require gross genomic alterations. Cell cycle (Georgetown, Tex.), 13(21), 3423-35.More infoTelomerase reactivation and immortalization are critical for human carcinoma progression. However, little is known about the mechanisms controlling this crucial step, due in part to the paucity of experimentally tractable model systems that can examine human epithelial cell immortalization as it might occur in vivo. We achieved efficient non-clonal immortalization of normal human mammary epithelial cells (HMEC) by directly targeting the 2 main senescence barriers encountered by cultured HMEC. The stress-associated stasis barrier was bypassed using shRNA to p16(INK4); replicative senescence due to critically shortened telomeres was bypassed in post-stasis HMEC by c-MYC transduction. Thus, 2 pathologically relevant oncogenic agents are sufficient to immortally transform normal HMEC. The resultant non-clonal immortalized lines exhibited normal karyotypes. Most human carcinomas contain genomically unstable cells, with widespread instability first observed in vivo in pre-malignant stages; in vitro, instability is seen as finite cells with critically shortened telomeres approach replicative senescence. Our results support our hypotheses that: (1) telomere-dysfunction induced genomic instability in pre-malignant finite cells may generate the errors required for telomerase reactivation and immortalization, as well as many additional "passenger" errors carried forward into resulting carcinomas; (2) genomic instability during cancer progression is needed to generate errors that overcome tumor suppressive barriers, but not required per se; bypassing the senescence barriers by direct targeting eliminated a need for genomic errors to generate immortalization. Achieving efficient HMEC immortalization, in the absence of "passenger" genomic errors, should facilitate examination of telomerase regulation during human carcinoma progression, and exploration of agents that could prevent immortalization.
- Rankin, T. M., Giovinco, N. A., Cucher, D. J., Watts, G., Hurwitz, B., & Armstrong, D. G. (2014). Three-dimensional printing surgical instruments: are we there yet?. The Journal of surgical research, 189(2), 193-7.More infoThe applications for rapid prototyping have expanded dramatically over the last 20 y. In recent years, additive manufacturing has been intensely investigated for surgical implants, tissue scaffolds, and organs. There is, however, scant literature to date that has investigated the viability of three-dimensional (3D) printing of surgical instruments.
- White, A. G., Watts, G. S., Lu, Z., Meza-Montenegro, M. M., Lutz, E. A., Harber, P., & Burgess, J. L. (2014). Environmental arsenic exposure and microbiota in induced sputum. International journal of environmental research and public health, 11(2), 2299-313.More infoArsenic exposure from drinking water is associated with adverse respiratory outcomes, but it is unknown whether arsenic affects pulmonary microbiota. This exploratory study assessed the effect of exposure to arsenic in drinking water on bacterial diversity in the respiratory tract of non-smokers. Induced sputum was collected from 10 subjects with moderate mean household water arsenic concentration (21.1 ± 6.4 ppb) and 10 subjects with low household water arsenic (2.4 ± 0.8 ppb). To assess microbiota in sputum, the V6 hypervariable region amplicons of bacterial 16s rRNA genes were sequenced using the Ion Torrent Personal Genome Machine. Microbial community differences between arsenic exposure groups were evaluated using QIIME and Metastats. A total of 3,920,441 sequence reads, ranging from 37,935 to 508,787 per sample for 316 chips after QIIME quality filtering, were taxonomically classified into 142 individual genera and five phyla. Firmicutes (22%), Proteobacteria (17%) and Bacteriodetes (12%) were the main phyla in all samples, with Neisseriaceae (15%), Prevotellaceae (12%) and Veillonellacea (7%) being most common at the genus level. Some genera, including Gemella, Lactobacillales, Streptococcus, Neisseria and Pasteurellaceae were elevated in the moderate arsenic exposure group, while Rothia, Prevotella, Prevotellaceae Fusobacterium and Neisseriaceae were decreased, although none of these differences was statistically significant. Future studies with more participants and a greater range of arsenic exposure are needed to further elucidate the effects of drinking water arsenic consumption on respiratory microbiota.
- Cyr, A. R., Brown, K. E., McCormick, M. L., Coleman, M. C., Case, A. J., Watts, G. S., Futscher, B. W., Spitz, D. R., & Domann, F. E. (2013). Maintenance of mitochondrial genomic integrity in the absence of manganese superoxide dismutase in mouse liver hepatocytes. Redox biology, 1, 172-7.More infoManganese superoxide dismutase, encoded by the Sod2 gene, is a ubiquitously expressed mitochondrial antioxidant enzyme that is essential for mammalian life. Mice born with constitutive genetic knockout of Sod2 do not survive the neonatal stage, which renders the longitudinal study of the biochemical and metabolic effects of Sod2 loss difficult. However, multiple studies have demonstrated that tissue-specific knockout of Sod2 in murine liver yields no observable gross pathology or injury to the mouse. We hypothesized that Sod2 loss may have sub-pathologic effects on liver biology, including the acquisition of reactive oxygen species-mediated mitochondrial DNA mutations. To evaluate this, we established and verified a hepatocyte-specific knockout of Sod2 in C57/B6 mice using Cre-LoxP recombination technology. We utilized deep sequencing to identify possible mutations in Sod2 (-/-) mitochondrial DNA as compared to wt, and both RT-PCR and traditional biochemical assays to evaluate baseline differences in redox-sensitive pathways in Sod2 (-/-) hepatocytes. Surprisingly, no mutations in Sod2 (-/-) mitochondrial DNA were detected despite measurable increases in dihydroethidium staining in situ and concomitant decreases in complex II activity indicative of elevated superoxide in the Sod2 (-/-) hepatocytes. In contrast, numerous compensatory alterations in gene expression were identified that suggest hepatocytes have a remarkable capacity to adapt and overcome the loss of Sod2 through transcriptional means. Taken together, these results suggest that murine hepatocytes have a large reserve capacity to cope with the presence of additional mitochondrial reactive oxygen species.
- Jandova, J., Beyer, T. E., Meuillet, E. J., & Watts, G. S. (2012). The matrix protein CCN1/CYR61 is required for α(V)β5-mediated cancer cell migration. Cell biochemistry and function, 30(8), 687-95.More infoCYR61 is one of the six proteins of the CCN family of proteins known to play diverse roles in angiogenesis, cellular proliferation, survival, migration and wound healing. However, the specific function of CYR61 in cancer is unclear, and the literature remains controversial. We used quantitative real-time PCR to establish the expression profile of CYR61 and integrin α(V)β5 in three non-small cell lung cancer, five colorectal cancer, one breast cancer and one oesophageal squamous carcinoma cell lines. We showed that the levels of CYR61 were significantly increased in oesophageal squamous carcinoma cell line along with the enhanced levels of α(V)β5 integrin. Further, we investigated whether tumour cell-secreted CYR61 can facilitate cell migration by interacting with the α(V)β5 integrin. Using tumour cell lines with low, intermediate and high CYR61 expression and their isogenic variants as a cellular model, we determined that integrin α(V)β5 expressed on these tumour cells is required for cell migration. Moreover, we showed that the modulation of expression levels of CYR61 in these cancer cells affected their capacity for migration. These results represent an advance to the understanding of the role of CYR61 and α(V)β5 integrin as proteins that cooperate to mediate cancer cell migration.
- Roesly, H. B., Khan, M. R., Chen, H. D., Hill, K. A., Narendran, N., Watts, G. S., Chen, X., & Dvorak, K. (2012). The decreased expression of Beclin-1 correlates with progression to esophageal adenocarcinoma: the role of deoxycholic acid. American journal of physiology. Gastrointestinal and liver physiology, 302(8), G864-72.More infoBeclin-1 has a central role in the regulation of autophagy. Barrett's esophagus (BE) is associated with a significantly increased risk for the development of esophageal adenocarcinoma (EAC). In the current study, we evaluated the role of Beclin-1 and autophagy in the EAC. Biopsies obtained from patients with BE and EAC, tissues from a rat model of BE and EAC, and esophageal cell lines were evaluated for the expression of Beclin-1 by immunohistochemistry, immunoblotting, or RT-PCR. Since reflux of bile acids is important in EAC, we also evaluated the effect of exposure to deoxycholic acid (DCA) on autophagy and Beclin-1 expression. Beclin-1 expression was high in squamous epithelium and nondysplastic BE, whereas its expression was low in dysplastic BE and EAC. The same pattern of expression was observed in rat tissues and in esophageal cell lines. Normal esophageal epithelium and HET-1A cells (derived from normal squamous epithelium) show high levels of Beclin-1, but lower levels of Beclin-1 were found in BE and EAC cell lines (CP-A, CP-C, and OE33). Acute exposure to DCA led to increased Beclin-1 expression and increased autophagy as evaluated by electron microscopy and counting percentage of GFP-LC3-positive BE cells with punctate pattern. In contrast, chronic exposure to DCA did not result in the alteration of Beclin-1 levels or autophagy. In summary, these data suggest that autophagy is initially activated in response to bile acids, but chronic exposure to bile acids leads to decreased Beclin-1 expression and autophagy resistance.
- Goldman, A., Shahidullah, M., Goldman, D., Khailova, L., Watts, G., Delamere, N., & Dvorak, K. (2010). A novel mechanism of acid and bile acid-induced DNA damage involving Na+/H+ exchanger: implication for Barrett's oesophagus. Gut, 59(12), 1606-16.More infoBarrett's oesophagus is a premalignant disease associated with oesophageal adenocarcinoma. The major goal of this study was to determine the mechanism responsible for bile acid-induced alteration in intracellular pH (pH(i)) and its effect on DNA damage in cells derived from normal oesophagus (HET1A) or Barrett's oesophagus (CP-A).
- Dvorak, K., Watts, G. S., Ramsey, L., Holubec, H., Payne, C. M., Bernstein, C., Jenkins, G. J., Sampliner, R. E., Prasad, A., Garewal, H. S., & Bernstein, H. (2009). Expression of bile acid transporting proteins in Barrett's esophagus and esophageal adenocarcinoma. The American journal of gastroenterology, 104(2), 302-9.More infoBarrett's esophagus (BE) is a metaplastic lesion characterized by replacement of the normal squamous epithelium by columnar intestinal epithelium containing goblet cells. It is speculated that this process is an adaptation to protect cells from components of refluxate, such as gastric acid and bile acids. In contrast to the normal squamous epithelium, enterocytes of the distal ileum are adapted to transport bile acids from the intestinal lumen. Several bile acid transporters are utilized for effective removal of bile acids, including the apical sodium-dependent bile acid transporter (ASBT), the ileal bile acid-binding protein (IBABP), and the multidrug-resistant protein 3 (MRP3). We hypothesized that one of the possible functions of newly arising metaplastic epithelium, in the esophagus, is to transport bile acids. Our major goal was to evaluate the expression of bile acid transporters in normal squamous epithelium, BE with different grades of dysplasia, and esophageal adenocarcinoma (EAC).
- 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.More infoAberrations 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.
- Novak, P., Jensen, T., Oshiro, M. M., Watts, G. S., Kim, C. J., & Futscher, B. W. (2008). Agglomerative epigenetic aberrations are a common event in human breast cancer. Cancer research, 68(20), 8616-25.More infoChanges in DNA methylation patterns are a common characteristic of cancer cells. Recent studies suggest that DNA methylation affects not only discrete genes, but it can also affect large chromosomal regions, potentially leading to LRES. It is unclear whether such long-range epigenetic events are relatively rare or frequent occurrences in cancer. Here, we use a high-resolution promoter tiling array approach to analyze DNA methylation in breast cancer specimens and normal breast tissue to address this question. We identified 3,506 cancer-specific differentially methylated regions (DMR) in human breast cancer with 2,033 being hypermethylation events and 1,473 hypomethylation events. Most of these DMRs are recurrent in breast cancer; 90% of the identified DMRs occurred in at least 33% of the samples. Interestingly, we found a nonrandom spatial distribution of aberrantly methylated regions across the genome that showed a tendency to concentrate in relatively small genomic regions. Such agglomerates of hypermethylated and hypomethylated DMRs spanned up to several hundred kilobases and were frequently found at gene family clusters. The hypermethylation events usually occurred in the proximity of the transcription start site in CpG island promoters, whereas hypomethylation events were frequently found in regions of segmental duplication. One example of a newly discovered agglomerate of hypermethylated DMRs associated with gene silencing in breast cancer that we examined in greater detail involved the protocadherin gene family clusters on chromosome 5 (PCDHA, PCDHB, and PCDHG). Taken together, our results suggest that agglomerative epigenetic aberrations are frequent events in human breast cancer.
- Watts, G. S., Futscher, B. W., Holtan, N., Degeest, K., Domann, F. E., & Rose, S. L. (2008). DNA methylation changes in ovarian cancer are cumulative with disease progression and identify tumor stage. BMC medical genomics, 1, 47.More infoHypermethylation of promoter CpG islands with associated loss of gene expression, and hypomethylation of CpG-rich repetitive elements that may destabilize the genome are common events in most, if not all, epithelial cancers.
- Goulet, A., Watts, G., Lord, J. L., & Nelson, M. A. (2007). Profiling of selenomethionine responsive genes in colon cancer by microarray analysis. Cancer biology & therapy, 6(4), 494-503.More infoHigh-selenium containing yeast is being evaluated in clinical trials against colon polyp recurrence. However, the molecular targets for the anticancer effects of selenium remain unclear. Previous studies by our group demonstrated that selenomethionine-induced growth arrest appears to be mediated by activation of ERK and subsequent phosphorylation of RSK and histone H3. These results suggest that selenomethionine can alter gene expression. In the present study, we have used cDNA microarrays to determine whether gene expression differences exist in HCT116 colon cancer cells treated with selenomethionine. These experiments reveal statistically significant expression changes for 50 genes. Genes we found to increase with selenomethionine treatment include KLK6, ATOX1, SGK, GJB2, DAP-1, PLAU, VIM, DPYSL2, STC2 and PXN. Conversely, genes downregulated by selenomethionine include PRKACB, LIM, DEPP, MYC, CDH5, ELF3, VSNL1, SAT and EGLN3. Further analysis of those genes using chromatin immunoprecipitation experiments showed that phosphorylated histone H3 on serine 10 bound to the GJB2 promoter (connexin 26) or the serum glucocorticoid kinase promoter is increased with selenomethionine treatment. Cells overexpressing CX26 or DAP-1 displayed a reduced number of colonies which suggests that these two genes could play a functional role in the growth inhibitory effects of selenomethionine. These data support the notion that selenomethionine-induced growth inhibition is associated with global changes in gene expression. They also demonstrate that selenomethionine can modify chromatin state to alter gene transcription. Finally, our studies provide a practical foundation for the further development of biomarkers to monitor the efficacy of selenomethionine in clinical trials.
- Watts, G. S., Tran, N. L., Berens, M. E., Bhattacharyya, A. K., Nelson, M. A., Montgomery, E. A., & Sampliner, R. E. (2007). Identification of Fn14/TWEAK receptor as a potential therapeutic target in esophageal adenocarcinoma. International journal of cancer. Journal international du cancer, 121(10), 2132-9.More infoGiven the poor survival rate and efficacy of current therapy for esophageal adenocarcinoma (EAC), there is a need to identify and develop new therapeutic targets for treatment. Microarray analysis (Affymetrix U133A GeneChips, Robust Multi-Chip Analysis) was used to expression profile 11 normal squamous and 18 Barrett's esophagus biopsies, 7 surgically resected EACs and 3 EAC cell lines. Two hundred transcripts representing potential therapeutic targets were identified using the following criteria: significant overexpression in EAC by analysis of variance (p = 0.05, Benjamini Hochberg false discovery rate); 3-fold increase in EAC relative to normal and Barrett's esophagus and expression in at least 2 of the 3 EAC cell lines. From the list of potential targets we selected TNFRSF12A/Fn14/TWEAK receptor, a tumor necrosis factor super-family receptor, for further validation based on its reported role in tumor cell survival and potential as a target for therapy. Fn14 protein expression was confirmed in SEG-1 and BIC-1 cell lines, but Fn14 was not found to affect tumor cell survival after exposure to chemotherapeutics as expected. Instead, a novel role in EAC was discovered in transwell assays, in which modulating Fn14 expression affected tumor cell invasion. Fn14's potential as a therapeutic target was further supported by immunohistochemistry on a tissue microarray of patient samples that showed that Fn14 protein expression increased with disease progression in EAC.
- Ignatenko, N. A., Yerushalmi, H. F., Watts, G. S., Futscher, B. W., Stringer, D. E., Marton, L. J., & Gerner, E. W. (2006). Pharmacogenomics of the polyamine analog 3,8,13,18-tetraaza-10,11-[(E)-1,2-cyclopropyl]eicosane tetrahydrochloride, CGC-11093, in the colon adenocarcinoma cell line HCT1161. Technology in cancer research & treatment, 5(6), 553-64.More infoPolyamine analogs are known to inhibit tumorigenesis at least in part by mimicking some of the regulatory roles of natural polyamines. To begin the identification of those signaling pathways that are involved in differential cellular responses to the synthetic conformationally restricted polyamine analog CGC-11093, we conducted gene expression profiling, proteomic, and genome-wide DNA methylation and histone acetylation analyses of the HCT116 colon adenocarcinoma cell line after treatment with this analog. Gene expression analysis was performed using Affymetrix GeneChip human genome U133 Plus 2.0 arrays. Changes in protein expression were evaluated using 2D polyacrylamide gels followed by LCMS/MS. DNA methylation was measured using 6,800 element CpG island microarrays. Treatment of cells with CGC-11093 at concentrations ranging from 0.1 to 10 microM caused inhibition of cell growth and metabolic activity, but only minimally affected cell viability. Gene expression analysis showed concentration-dependent effects of CGC-11093 on the DNA/RNA binding transcription factor, cell cycle, signaling, transport, cytoskeletal/structural, and serine protease genes. Functional gene analysis revealed distinct expression patterns related to inhibition of cell cycle control, TGF beta signaling, proteasome and RNA polymerase pathways, upregulation of the aminoacyl-tRNA synthesis pathway, and perturbations in the MAPK and Wnt signaling pathways. Microarray results were validated for selected genes with real time RT PCR. Proteomics analysis showed correlative changes in the expression of proteins involved in the regulation of proteasome function (proteasome subunit Y) and tRNA synthesis. CGC-11093 treatment did not produce any detectable changes in DNA methylation or histone acetylation in cells. This study validates specific target pathways for a specific conformationally restricted polyamine analog and suggests the utility of combined gene and DNA methylation microarrays along with proteomic analyses as a useful approach to the evaluation of the mechanisms of action of anticancer drugs.
- Li, H., Watts, G. S., Oshiro, M. M., Futscher, B. W., & Domann, F. E. (2006). AP-2alpha and AP-2gamma are transcriptional targets of p53 in human breast carcinoma cells. Oncogene, 25(39), 5405-15.More infoActivating enhancer-binding protein 2alpha (AP-2alpha) and activating enhancer-binding protein 2gamma (AP-2gamma) are transcription factors that bind GC-rich consensus sequences and regulate the expression of many downstream genes. AP-2alpha and AP-2gamma interact with p53 both physically and functionally. Expression microarray results in human breast carcinoma cells with forced p53 expression revealed AP-2gamma as a putative transcriptional target of p53. To confirm and extend these findings we measured the effects of forced p53 expression in human breast carcinoma cells by real-time reverse transcription-PCR, Western blotting, electrophoretic gel mobility shift assays, promoter reporter, chromatin immunoprecipitation and chromatin accessibility assays. Wild-type p53 expression rapidly induced not only AP-2gamma but also AP-2alpha mRNA. The subsequent increase in these proteins led to increased AP-2 DNA-binding and transactivating activity. Candidate p53-binding sites were identified in the AP-2alpha and AP-2gamma promoters. p53 binding to these cis-elements in vivo was also observed, together with a relaxation of chromatin structure in these regions. Finally, expression of either AP-2alpha or gamma inhibited growth of human breast carcinoma cells in vitro. Taken together, our findings indicate that these AP-2 genes are targets for transcriptional activation by p53 and suggest that AP-2 proteins may mediate some of the downstream effects of p53 expression such as inhibition of proliferation.
- Novak, P., Jensen, T., Oshiro, M. M., Wozniak, R. J., Nouzova, M., Watts, G. S., Klimecki, W. T., Kim, C., & Futscher, B. W. (2006). Epigenetic inactivation of the HOXA gene cluster in breast cancer. Cancer research, 66(22), 10664-70.More infoUsing an integrated approach of epigenomic scanning and gene expression profiling, we found aberrant methylation and epigenetic silencing of a small neighborhood of contiguous genes-the HOXA gene cluster in human breast cancer. The observed transcriptional repression was localized to approximately 100 kb of the HOXA gene cluster and did not extend to genes located upstream or downstream of the cluster. Bisulfite sequencing, chromatin immunoprecipitation, and quantitative reverse transcription-PCR analysis confirmed that the loss of expression of the HOXA gene cluster in human breast cancer is closely linked to aberrant DNA methylation and loss of permissive histone modifications in the region. Pharmacologic manipulations showed the importance of these aberrant epigenetic changes in gene silencing and support the hypothesis that aberrant DNA methylation is dominant to histone hypoacetylation. Overall, these data suggest that inactivation of the HOXA gene cluster in breast cancer may represent a new type of genomic lesion-epigenetic microdeletion. We predict that epigenetic microdeletions are common in human cancer and that they functionally resemble genetic microdeletions but are defined by epigenetic inactivation and transcriptional silencing of a relatively small set of contiguous genes along a chromosome, and that this type of genomic lesion is metastable and reversible in a classic epigenetic fashion.
- Ignatenko, N. A., Zhang, H., Watts, G. S., Skovan, B. A., Stringer, D. E., & Gerner, E. W. (2004). The chemopreventive agent alpha-difluoromethylornithine blocks Ki-ras-dependent tumor formation and specific gene expression in Caco-2 cells. Molecular carcinogenesis, 39(4), 221-33.More infoMutation of the Kirsten-ras (Ki-ras) proto-oncogene occurs frequently in colorectal cancers. alpha-Difluoromethylornithine (DFMO), an irreversible inhibitor of the polyamine biosynthetic enzyme, ornithine decarboxylase (ODC), inhibits Ki-ras transformation and colon tumorigenesis in carcinogen-treated animal models by mechanisms yet to be elucidated. Caco-2 cells transfected with an activated Ki-ras, but not parental cells, formed tumors in severe combined immunodeficient (SCID) mice. DFMO treatment (2% in drinking water) prevented tumor growth. Gene expression profiling was performed to identify Ki-ras-and DFMO-dependent patterns of gene expression. Microarray results were validated with real-time or semi-quantitative RT-PCR and/or Western blot analysis. Genes upregulated in Caco-2 cells expressing an activated Ki-ras encoded cytoskeletal-, transport-, protease-, and gap junction-associated proteins. These genes are important for normal development and maintenance of colonic epithelial tissue. Caco-2 cells transfected with an activated Ki-ras displayed increased expression of the integrin alpha 1 (INGA1) and enhanced cell migration on laminin. These parameters were unaffected by DFMO, but Ki-ras-dependent migration was inhibited by INGA1 antibodies. Other Ki-ras-dependent, but DFMO-independent, genes included transglutaminase (TGase) and kallikrein 6 (KLK6). Ki-ras-transfected cells also expressed increased levels of connexin43 (Cx43) (RNA and protein), tight junction protein, and endothelin 1. DFMO reversed these increases. The results indicated that the Ki-ras oncogene caused changes in experimental cell migration and cell-cell communication genes and that some of these changes could be reversed by DFMO.
- Shippy, R., Sendera, T. J., Lockner, R., Palaniappan, C., Kaysser-Kranich, T., Watts, G., & Alsobrook, J. (2004). Performance evaluation of commercial short-oligonucleotide microarrays and the impact of noise in making cross-platform correlations. BMC genomics, 5, 61.More infoDespite the widespread use of microarrays, much ambiguity regarding data analysis, interpretation and correlation of the different technologies exists. There is a considerable amount of interest in correlating results obtained between different microarray platforms. To date, only a few cross-platform evaluations have been published and unfortunately, no guidelines have been established on the best methods of making such correlations. To address this issue we conducted a thorough evaluation of two commercial microarray platforms to determine an appropriate methodology for making cross-platform correlations.
- Oshiro, M. M., Watts, G. S., Wozniak, R. J., Junk, D. J., Munoz-Rodriguez, J. L., Domann, F. E., & Futscher, B. W. (2003). Mutant p53 and aberrant cytosine methylation cooperate to silence gene expression. Oncogene, 22(23), 3624-34.More infop53 is an important transcriptional regulator that is frequently mutated in cancer. Gene-profiling experiments of breast cancer cells infected with wt p53 revealed both MASPIN and desmocollin 3 (DSC3) to be p53-target genes, even though both genes are silenced in association with aberrant cytosine methylation of their promoters. Despite the transcriptional repression of these genes by aberrant DNA methylation, restoration of p53 resulted in the partial reactivation of both genes. This reactivation is a result of wt p53 binding to its consensus DNA-binding sites within the MASPIN and DSC3 promoters, stimulating histone acetylation, and enhancing chromatin accessibility of their promoters. Interestingly, wt p53 alone did not affect the methylation status of either promoter, suggesting that p53 itself can partially overcome the repressive barrier of DNA methylation. Pharmacologic inhibition of DNA methylation with 5-aza-2'-deoxycytidine in combination with restoration of wt p53 status resulted in a synergistic reactivation of these genes to near-normal levels. These results suggest that cancer treatments that target both genetic and epigenetic facets of gene regulation may be a useful strategy towards the therapeutic transcriptional reprogramming of cancer cells.
- Zheng, X. H., Watts, G. S., Vaught, S., & Gandolfi, A. J. (2003). Low-level arsenite induced gene expression in HEK293 cells. Toxicology, 187(1), 39-48.More infoChronic, low-level exposure to arsenic frequently results in skin, lung, bladder, and kidney cancer. Since arsenic is primarily excreted via the kidney, this study focused on this target tissue. Gene array was used as a sensitive low-level monitor of the impact of arsenic on this target tissue. Arsenite [As(III)] was chosen as the chemical species of arsenic since As(III) species are touted as the cellular toxic form of arsenic. Human embryonic kidney cell line HEK293 cells were incubated with 1, 10, and 25 microM arsenite [As(III)] for 6 or 24 h. Total RNA from treated and control cells was isolated, reverse transcribed, and labeled with Cy3 or Cy5, and hybridized to a human cDNA microarray. Hybridizations were performed four times using independent total RNA preparations to ensure reproducibility. Raw data from 10 and 25 microM treated cells exposed for 6 h was normalized within, and between, hybridizations followed by identification of genes affected by arsenite exposure based on practical significance (2-fold change up or down) and reproducibility (affected in four of six measurements). In these studies, 20 genes (HMOX1, MT1E, or FOSL1, etc.) were up-regulated, and 19 genes (MYC, JAK1, or CENPE, etc.) were down-regulated. Genes identified at 10 and 25 microM arsenic exposure were then examined after 1 microM treatment for 6 or 24 h. Expression of affected genes showed a dose-dependent (1-25 microM) trend that was apparently not time-dependent (6 vs. 24 h). The affected genes indicate that even this realistic, low-level arsenite exposure was recognized by the HEK293 cells (e.g. metallothionein genes) and produced an oxidative stress (e.g. heme oxygenase gene). These affected genes were characterized as stress response genes, proto-oncogene, signaling molecules, transcription factors, chemokine receptors, proteolytic enzymes, ESTs, and unknown genes. These findings imply that arsenite induces complex cellular injury and the cellular adaptation to As(III) is associated with alterations in the expression of many genes.
- Crowley-Weber, C. L., Payne, C. M., Gleason-Guzman, M., Watts, G. S., Futscher, B., Waltmire, C. N., Crowley, C., Dvorakova, K., Bernstein, C., Craven, M., Garewal, H., & Bernstein, H. (2002). Development and molecular characterization of HCT-116 cell lines resistant to the tumor promoter and multiple stress-inducer, deoxycholate. Carcinogenesis, 23(12), 2063-80.More infoEvidence from live cell bioassays shows that the flat mucosa from patients with colon cancer exhibits resistance to bile salt-induced apoptosis. Three independent cell lines derived from the colonic epithelial cell line HCT-116 were selected for resistance to bile salt-induced apoptosis. These cell lines were developed as tissue culture models of apoptosis resistance. Selection was carried out for resistance to apoptosis induced by sodium deoxycholate (NaDOC), the bile salt found in highest concentrations in human fecal water. Cultures of HCT-116 cells were serially passaged in the presence of increasing concentrations of NaDOC. The resulting apoptosis resistant cells were able to grow at concentrations of NaDOC (0.5 mM) that cause apoptosis in a few hours in unselected HCT-116 cells. These cells were then analyzed for changes in gene expression. Observations from cDNA microarray, 2-D gel electrophoresis/MALDI-mass spectroscopy, and confocal microscopy of immunofluorescently stained preparations indicated underexpression or overexpression of numerous genes at either the protein or mRNA level. Genes that may play a role in apoptosis and early stage carcinogenesis have been identified as upregulated in these cell lines, including Grp78, Bcl-2, NF-kappaB(p50), NF-kappaB(p65), thioredoxin peroxidase (peroxiredoxin) 2, peroxiredoxin 4, maspin, guanylate cyclase activating protein-1, PKCzeta, EGFR, Ras family members, PKA, PI(4,5)K, TRAF2 and BIRC1 (IAP protein). Under-expressed mRNAs included BNIP3, caspase-6, caspase-3 and serine protease 11. NF-kappaB was constitutively activated in all three resistant cell lines, and was responsible, in part, for the observed apoptosis resistance, determined using antisense oligonucleotide strategies. Molecular and cellular analyses of these resistant cell lines has suggested potential mechanisms by which apoptosis resistance may develop in the colonic epithelium in response to high concentrations of hydrophobic bile acids that are associated with a Western-style diet. These analyses provide the rationale for the development of hypothesis-driven intermediate biomarkers to assess colon cancer risk on an individual basis.
- Calaluce, R., Kunkel, M. W., Watts, G. S., Schmelz, M., Hao, J., Barrera, J., Gleason-Guzman, M., Isett, R., Fitchmun, M., Bowden, G. T., Cress, A. E., Futscher, B. W., & Nagle, R. B. (2001). Laminin-5-mediated gene expression in human prostate carcinoma cells. Molecular carcinogenesis, 30(2), 119-29.More infoInteractions between extracellular matrix (ECM) proteins and prostate carcinoma cells provide a dynamic model of prostate tumor progression. Previous work in our laboratory showed that laminin-5, an important member of a family of ECM glycoproteins expressed in the basal lamina, is lost in prostate carcinoma. Moreover, we showed that the receptor for laminin-5, the alpha6beta4 integrin, is altered in prostate tumors. However, the genes that laminin-5 potentially regulates and the significance of its loss of expression in prostate cancer are not known. We selected cDNA microarray as a comprehensive and systematic method for surveying and examining gene expression induced by laminin-5. To establish a definitive role for laminin-5 in prostate tumor progression and understand the significance of its loss of expression, we used a cDNA microarray containing 5289 human genes to detect perturbations of gene expression when DU145 prostate carcinoma cells interacted with purified laminin-5 after 0.5, 6, and 24 h. Triplicate experiments showed modulations of four, 61, and 14 genes at 0.5, 6, and 24 h, respectively. Genes associated with signal transduction, cell adhesion, the cell cycle, and cell structure were identified and validated by northern blot analysis. Protein expression was further assessed by immunohistochemistry. Mol. Carcinog. 30:119-129, 2001.
- Watts, G. S., Futscher, B. W., Isett, R., Gleason-Guzman, M., Kunkel, M. W., & Salmon, S. E. (2001). cDNA microarray analysis of multidrug resistance: doxorubicin selection produces multiple defects in apoptosis signaling pathways. The Journal of pharmacology and experimental therapeutics, 299(2), 434-41.More infoDoxorubicin plays an important role in the treatment of leukemias, lymphomas, and a variety of carcinomas. Tumor cell resistance to doxorubicin is often associated with expression of the multidrug resistance gene MDR1, which codes for the drug efflux pump P-glycoprotein, and a multidrug-resistant phenotype. Evidence from multiple sources suggests, however, that additional genes besides MDR1 are involved in development of multidrug resistance. To identify genes involved in the multidrug resistance phenotype, we created a 5760-gene cDNA microarray to search for differentially expressed genes between the human multiple myeloma cell line RPMI 8226 and its doxorubicin-selected sublines 8226/Dox6 and 8226/Dox40, both of which express MDR1 and are multidrug-resistant. The cDNA microarray results identified a set of differentially expressed genes, which included MDR1 as expected. Thirty Northern analyses were used to confirm the results of the cDNA microarrays; comparison with the microarray results showed a 90% agreement between the two techniques. Within the set of differentially expressed genes identified by the cDNA microarrays, 29 were of particular interest as they can participate in apoptotic signaling, particularly as mediated by ceramide and the mitochondrial permeability transition. The functional importance of these changes in gene expression is supported by their explanation of the 8226/Dox cell lines' cross-resistance to substances that are not P-glycoprotein substrates, such as Fas/CD95 ligand and staurosporine. We conclude that doxorubicin selection led to changes in gene expression that reduce the apoptotic response to death-inducing stimuli and thus contribute to the multidrug resistance phenotype.
- Watts, G. S., Pieper, R. O., Costello, J. F., Peng, Y. M., Dalton, W. S., & Futscher, B. W. (1997). Methylation of discrete regions of the O6-methylguanine DNA methyltransferase (MGMT) CpG island is associated with heterochromatinization of the MGMT transcription start site and silencing of the gene. Molecular and cellular biology, 17(9), 5612-9.More infoO6-Methylguanine DNA methyltransferase (MGMT) repairs the mutagenic and cytotoxic O6-alkylguanine lesions produced by environmental carcinogens and the chemotherapeutic nitrosoureas. As such, MGMT-mediated repair of O6-alkylguanine lesions constitutes a major form of resistance to nitrosourea chemotherapy and makes control of MGMT expression of clinical interest. The variability of expression in cell lines and tissues, along with the ease with which the MGMT phenotype reverts under various conditions, suggests that MGMT is under epigenetic control. One such epigenetic mechanism, 5-methylation of cytosines, has been linked to MGMT expression. We have used an isogenic human multiple myeloma tumor cell line model composed of an MGMT-positive parent cell line, RPMI 8226/S, and its MGMT-negative variant, termed 8226/V, to study the control of MGMT expression. The loss of MGMT activity in 8226/V was found to be due to the loss of detectable MGMT gene expression. Bisulfite sequencing of the MGMT CpG island promoter revealed large increases in the levels of CpG methylation within discrete regions of the 8226/V MGMT CpG island compared to those in 8226/S. These changes in CpG methylation are associated with local heterochromatinization of the 8226/V MGMT transcription start site and provide a likely mechanism for the loss of MGMT transcription in 8226/V.
- Watts, G. S., & Futscher, B. W. (1995). Detecting differences in 5-methylcytosine using restriction enzyme isoschizomers: an endogenous control for complete digestion. Nucleic acids research, 23(22), 4740-1.