Cachexia is the most debilitating syndrome which manifests itself in several chronic, life-threatening diseases, especially in cancer. Cachexia is of major public health significance for the cancer population because it increases both morbidity and mortality and also reduces quality of life and survival time of cancer patients. Up to two-thirds of patients with advanced neoplasia develop signs and symptoms of cachexia, including anorexia, asthenia and severe unintentional weight loss leading to immobility and cardiac or respiratory failure. Cachexia accounts for more than 20% of all cancer-associated deaths. In the present study, we established a novel murine model for cancer cachexia induced by the human prostate cancer cell line PC-3. In a novel mouse model of cachexia induced by PC-3 cells, an established androgen-independent cell line derived from a bone metastasis of a human prostatic adenocarcinoma, in BALB/c nude mice we observed body weight loss, a 50% reduction in muscle weights and decreased muscle fiber diameters. Elevated levels of the phosphorylated p65 subunit of the nuclear factor of êB (NF-êB) were found in tibialis anterior and quadriceps muscles, but not in gastrocnemius muscle. Elevated levels of the muscle specific E3 ligase (MuRF1) confirmed activation of the ubiquitin-proteosome protein degradation pathway in these muscles. In contrast, elevated levels of the phosphorylated eukaryotic initiation factor 2 alpha (p-eIF2-á) in gastrocnemius muscle, but not in tibialis anterior and quadriceps muscles, suggested a greater component of cachexia due to decreased protein synthesis in this muscle. We also utilized a well-established murine model of cancer cachexia induced by murine colon adenocarcinoma cell line (C-26). C-26 tumor-bearing mice were treated with an intramuscular injection of an adeno-associated viral vector serotype 8 (AAV8) carrying the IêB super repressor (IêBSR) or cellular caspase-8-like inhibitory protein (cFLIP) gene driven by the cytomegalovirus (CMV) or muscle creatine kinase (MCK) promoter. We found that there was an improvement in body weight, individual muscle weight and muscle fiber diameter in mice receiving AAV8-IêBSR or AAV8-cFLIP. We also observed a reduction of MuRF1 protein expression, indicating that there was a reduction in muscle protein degradation via the ubiquitin-proteasome system. The result was confirmed by an increased level of myosin heavy chain protein expression. This study suggests the potential for AAV8 carrying IBSR gene mediated gene transfer to prevent or reverse cachectic symptoms in vivo.

Smoking is one of the leading risk factors that contribute to the development of head and neck squamous cell carcinoma (HNSCC). Smoking induces chromosome breaks. One of the most frequent and earliest alterations in HNSCC is the segmental loss of the short arm of chromosome 3 (3p). Another frequent observation is the activation of one of the most common fragile sites in HNSCC, FRA3B, which spans the FHIT gene, considered to be a tumor suppressor. Distal to FHIT is FANCD2, which encodes a protein required for the proper function of the Fanconi anemia/BRCA pathway and eventual repair of DNA damage through homologous recombination repair. Recently, it has been observed that FANCD2 gene and protein expression is decreased in HNSCC cells. Therefore, we hypothesize that 3p loss, resulting from the activation of the fragile site FRA3B, leads to FANCD2 gene copy loss and a defective DNA damage response. Western blots showed decreased expression of FHIT protein in HNSCC cell lines. Fluorescence in situ hybridization revealed partial loss of FANCD2 in HNSCC cell lines studied. Western blotting and quantitative RT-PCR showed decreased FANCD2 protein and gene expression in HNSCC cell lines with FANCD2 gene copy loss. HNSCC cell lines with decreased FHIT protein expression, FANCD2 gene copy loss, and decreased FANCD2 gene and protein expression exhibited decreased FANCD2 and RAD51 focus formation. Our results suggest that smoking could induce breakage of the fragile site, FRA3B, leading to FANCD2 loss and resulting in defective DNA damage repair. The use of targeted therapy on cancer cells with deficient DNA damage repair, like CHEK1 small molecule inhibitors, could improve the currently available cancer treatment schemes. The public health relevance of our studies involves the use of an abnormal FA/BRCA pathway as a marker for selective use of targeted cancer therapy for HNSCC.

Triploidy is the presence of 69 chromosomes instead of the normal diploid number of 46 and can occur in a complete form or in a mixoploid state in which there are populations of diploid and triploid cells in the same individual. The extra haploid set can be of paternal or maternal origin. Triploidy is one of the most common chromosome aberrations seen in 1-2% of all recognized pregnancies and can lead to partial mole which can in turn lead to serious complications for the mother and fetus. Given the high incidence of chromosome abnormalities including triploidy and its impact on individuals with chromosomally abnormal pregnancies, a greater understanding of their etiology has a potential to contribute greatly to public health by enhancing the management and possible future prevention. Though complete triploidy is not compatible with postnatal survival, mixoploid individuals are capable of surviving into adulthood. Both syndromes have a broad phenotypic spectrum though it is generally less severe in mixoploids. Though much has been learned in the nearly half century since the first case report of diploid/triploid mixoploidy was published, many questions still remain. A major issue is a large between study difference in the ratio of diandric to digynic triploidy and the prevalence of partial hydatidiform mole. Additionally, there is a clear parent-of-origin effect on fetal and placental morphology as well as developmental age that is believed to be related to genomic imprinting. The goals of this paper include summarizing the current body of knowledge on triploidy and diploid/triploid mixoploidy, examining the remaining questions, and a side-by-side comparison of the two syndromes. An exhaustive literature search was undertaken which produced many case reports of triploidy and diploid/triploid mixoploidy as well as studies on the mechanisms leading to triploidy, phenotypic characteristics, and the characteristics of triploid cells. It appears that the complex pattern surrounding parental origin of the extra haploid set of chromosomes may have contributed to between study ascertainment bias. More complex studies with careful attention to detail must be undertaken to fully understand the etiology and pathophysiology of triploidy.

Background: Ischemic heart disease remains the number one killer in the developed world. A protein transduction peptide specific for the heart capable of efficiently delivering agents of therapeutic potential at the time of injury, would be of immense public health significance. This work was undertaken to identify peptide(s) able to transduce heart tissue in vivo in a tissue-specific manner. Biopanning was performed in cell culture followed by in vivo with an M13 phage peptide display library. Using the heart-specific peptide, we delivered nemo-binding domain peptide in a murine infarct model to test if NF-kB inhibition can reduce infarct size. Methods and Results: A cardiomyoblast cell line, H9C2, was incubated with M13 twelve amino acid phage peptide display library. Internalized phage was recovered, amplified and subjected to a total of three rounds of in vivo biopanning where infectious, internalized phage was isolated from cardiac tissue following intravenous injection. After the third round, 60% of sequenced plaques carried the peptide sequence APWHLSSQYSRT, termed cardiac targeting peptide (CTP). This peptide was synthesized either fluorescently labeled, biotinylated, or in combination with a NEMO-binding peptide (NBD), an inhibitor of the inducible NF-kappa B Kinase (IKK). We demonstrate that CTP was able to transduce cardiomyocytes functionally in culture in a concentration and cell-type dependent manner. Mice injected with CTP showed significant transduction of heart tissue with minimal uptake by lung and kidney capillaries, and no uptake in liver, skeletal muscle, spleen or brain. The level of heart transduction by CTP also was not observed with a cationic transduction domain. CTP-NBD was able to inhibit NF-kB activation in cell culture in a dose-dependent fashion. When administered to mice in a murine infarct model, CTP-NBD showed a trend towards smaller infarct size, which did not reach statistical significance. Conclusions: Biopanning using a peptide phage display library identified a peptide, termed CTP, able to transduce cardiomyoblast cell line in vitro, and heart tissue in vivo, efficiently and specifically. Administration of CTP-NBD to post-infarct mice showed a trend towards reduction of infarct size. CTP could be used to deliver therapeutic peptides, proteins and nucleic acid specifically to the heart.

ERCC1-XPF is a mammalian structure specific endonuclease that incises at the junction of double and 3 single-stranded DNA. ERCC1-XPF and its homologs are implicated in multiple DNA repair pathways including, nucleotide excision repair, interstrand crosslink repair, double strand break repair, repair of AP sites, and telomere maintenance, making it essential for viability. The pleiotropic phenotypes of ERCC1-XPF deficiency in humans, range from the cancer-predisposition syndrome xeroderma pigmentosum to the XPF-ERCC1 progeroid syndrome, a disease of accelerated aging. This document concentrates on dissecting the role of ERCC1-XPF in crosslink repair, elucidating the mechanisms underlying the dramatic phenotypic differences in patients with inherited XPF mutations, and exploring the utility of ERCC1 and XPF as biomarkers of tumor prognosis. Ercc1 and Xpf knockout mice are phenocopies, illustrating that the two proteins function exclusively as a heterodimer. These models present a progeroid phenotype, reflected in some patients with XPF or ERCC1 mutations. We hypothesize that this accelerated aging is consequent on a failure to repair crosslinks, which are highly cytotoxic DNA lesions. The crosslink repair mechanism in animals remains poorly elucidated and the exact relationship of ERCC1-XPF to other players in this pathway, largely unexplored. Herein we show that ERCC1-XPF functions in the same pathway of crosslink repair as the Fanconi anemia proteins and that crosslink unhooking by ERCC1-XPF is required for the efficient binding of FANCD2 to the chromatin. The pleiotropy of phenotypes associated with human ERCC1-XPF deficiency is puzzling. Often, the severity of phenotype does not correlate with the DNA incising ability of the mutant proteins. We show here that the phenotypic heterogeneity is at least partially explained by the mislocalization of mutant proteins to the cytoplasm. The public health importance of this work comes from the increasing interest in the regulation of ERCC1-XPF expression and activity, due to its involvement in DNA repair pathways implicated in the resistance of tumors to platinum-based chemotherapy. A panel of antibodies was tested extensively and optimized for use in clinical measurement of ERCC1-XPF. This will facilitate improved measurement of DNA repair proteins in clinical specimens and greater understanding of the mechanisms of tumor resistance to genotoxic therapy.

E47, an alternative splice product of the transcription factor 3 (TCF3) gene, has been mechanistically linked with multiple leukemias and lymphomas, and thus it is of great public health importance to study the mechanisms by which E47 influences the development of the hematopoietic system. Throughout life, all mature blood cells are constantly replenished from rare, self-renewing bone marrow hematopoietic stem cells (HSCs) and downstream non-renewing multipotent progenitors (MPPs). Little is known about the gene regulatory network that controls the integrity of these essential bone marrow subsets. Previous evidence has suggested a crucial role for the transcription factor E47 in lymphocyte lineage commitment. However, the specific stages of hematopoiesis that require E47 and the underlying mechanisms through which it acts on remain unclear. Our study aims to elucidate the role of the transcription factor E47 in the earliest, multipotent stages of hematopoiesis. Using E47 deficient mice, we found that E47 is required for the development and functional integrity of uncommitted hematopoietic progenitors. Our results showed that E47 deficient mice had a 5070% reduction in non-renewing MPPs, and the residual MPPs failed to initiate V(D)J recombination, a hallmark of lymphoid lineage progression. The long-term lineage repopulation and self-renewal activities of the primitive HSCs are also compromised in the absence of E47. Not only were the in vivo long-term repopulating HSCs reduced by 3 fold in the bone marrow of E47 deficient mice, but also these HSCs displayed poor self-renewal efficiency by serial transplantation. The compromised self-renewal of E47 null HSCs appears to be associated with premature exhaustion due to over-proliferation under replication stress. The multipotent hematopoietic stem/progenitor cells from E47 deficient mice displayed a striking hyperproliferation following transplantation stress, and they exhibited increased susceptibility to in vivo challenge with a mitotoxic drug. Finally, loss of function and gain of function assays identified the cell cycle inhibitor p21 as a target gene of E47. Together, these observations suggested that E47 regulates the development and functional potential of multipotent hematopoietic subsets, probably through effects on p21-mediated cell cycle quiescence. These findings might provide novel mechanistic insights into hematopoietic damage repair and malignant transformation.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and usually lethal fibrotic lung disease with as yet unknown etiology and treatment. IPF lungs have a definitive gene expression signature, but the role of microRNAs in IPF has not been studied. MicroRNA genes are short, non-coding RNAs that function as post-transcriptional gene regulators. We hypothesized that microRNAs are differentially expressed in IPF and play a role in the pathogenesis of the disease. The microRNA expression profile of IPF lungs was characterized using microRNA microarrays. We identified 18 significantly decreased and 28 significantly increased microRNAs in samples obtained from patients with IPF compared to controls. Promoter analysis of the decreased microRNAs recognized a SMAD3 binding site in the promoter of let-7d. SMAD3 binding and responsiveness to TGF-β of the let-7d promoter were confirmed by SMAD3 ChIP, EMSA, luciferase assays and reduced expression of let-7d in response to TGF-β. In situ hybridization confirmed a significant reduction in let-7d expression in IPF lungs and localized it to alveolar epithelial cells in the control lungs. To study the role of let-7d, we determined the consequences of loss of function of let-7d by inhibiting its expression in lung epithelial cells. In-vitro inhibition of let-7d caused increases in epithelial mesenchymal transition (EMT) markers N-cadherin (CDH2), vimentin (VIM), alpha-smooth muscle actin (ACTA2) and high mobility group AT-hook 2 (HMGA2). HMGA2 is a proven let-7d target that was increased in IPF lungs and localized to alveolar epithelial cells. We reproduced our in vitro results in vivo. In mice, intratracheal administration of a let-7d inhibitor caused alveolar septal thickening, increases in collagen, ACTA2 and CDH2 and decreases in the epithelial markers, CDH1 and ZO1. We colocalized the mesenchymal markers FSP1 and ACTA2 with the epithelial marker SPC, indicative of EMT. Our results indicate a definitive role for microRNAs in IPF. The downregulation of let-7d in IPF and the pro-fibrotic effects of this downregulation in vitro and in vivo suggest a key regulatory role for this microRNA in preventing lung fibrosis. Deciphering their mechanism of action of microRNAs has great public health significance. It would add new insight into the pathophysiology of the disease and aid in reducing the mortality by development of therapeutic interventions.

Prostate cancer (PC) remains a significant public health concern for men throughout the world. Identification of the environmental and genetic factors that predispose to PC for prevention or early intervention is a significant public health concern. PC is more prevalent in males of African descent living in the western hemisphere. Also, recent studies have shown high rates of PC in Afro-Caribbean populations. Men in the 40-79 year age range from the Caribbean island of Tobago have a 3 fold higher risk of developing prostate cancer than do Caucasians. We performed two candidate gene-based association studies to explore the genetic determinants for high risk of PC in Tobago population. In the first study, we performed an extensive study to identify sequence variation in the DC-SIGN gene, a candidate gene for PC, in the Tobago population, and carried out a case/control association study of DC-SIGN polymorphisms and PC susceptibility in the high-risk population of Tobago. We found a unique haplotype in the 5 proximal promoter of DC-SIGN that is associated with PC in Tobago. We further extended the association study to single gene polymorphisms likely to be in linkage disequilibrium with DC-SIGN. We also found that a single nucleotide polymorphism, rs4804806, which is located in the intragenic region between the DC-SIGN and DCSIGN-R genes (4228bp upstream of DC-SIGN), is significantly associated with elevated risk of PC in Tobago population. The function of rs4804806, and whether the risk associated with it is due to linkage disequilibrium between it and other causal variation remains to be explored. In the second study, we examined whether genetic variation in the androgen receptor (AR) and PSA genes was associated with risk of prostate cancer or with serum PSA levels in the Tobago population. Previous studies have proposed that the rs266882 G/A polymorphism, located in the androgen response element upstream of the PSA gene and the (CAG)n repeat polymorphism in DNA binding domain of the AR are associated with risk of PC and serum PSA levels, and those two genetic variants show evidence of genetic interaction. In order to examine whether those two genetic variants affect PC risk or PSA level in Tobago population, an association study was carried out in 167 PC cases and 320 controls. Association analysis with PC was conducted among all cases and controls for each locus individually, and also for both loci together, to test for interaction between them in determining risk of PC or PSA levels. Regression analysis was carried out for each locus and combined with serum PSA level in 320 controls only. Results from this study do not provide any association evidence with PC risk or PSA level for PSA rs266882 G/A genotypes or AR (CAG)n repeat length genotypes. The results from these two studies lend support to exploring genetic causes and prevention of prostate cancer.

Objectives: Mammographic breast density (MBD) is a strong risk factor for breast cancer. It has been hypothesized that breast cancer susceptibility loci may also be associated with breast density. Recently, two genome-wide association studies identified a single-nucleotide polymorphism (SNP), rs2981582, in intron 2 of the FGFR2 gene to be associated with increased breast cancer risk. Further research revealed that intron 2 of FGFR2 contains estrogen receptor transcription factor binding sites. We examined associations of four FGFR2 SNPs (rs2981582, rs3750817, rs17542768 and rs1219643), hormone therapy (HT) use, and their interactions with MBD. Methods: We conducted a cross-sectional analysis using a subset of the Mammograms and Masses Study population. Subjects were 370 healthy postmenopausal Caucasian women. General linear models adjusted for covariates were used to evaluate the associations. Results: Overall, no statistically significant associations were observed between the four SNPs in FGFR2 and MBD. Duration of estrogen plus progestin use, but not duration of estrogen use, and HT status were statistically significantly associated with MBD in our study population. No statistically significant interactions between genotypes and HT use were observed. Conclusions: Our results suggest that the effects of the four evaluated FGFR2 polymorphisms on breast cancer risk are not mediated through MBD, and that the polymorphisms do not modify the effect of HT use on MBD. Implications for public health: Breast cancer is the most common cancer in women in the U.S. Identification of genes that influence MBD may provide insight into the biology of breast density and its effect on breast cancer, eventually leading to more effective prevention and treatment.

The incidence and mortality of brain tumors have not changed over the last 3 decades and pose a significant burden on the healthcare system of the United States. Temozolomide (TMZ) is the preferred chemotherapeutic agent for the treatment of brain tumors and base excision repair (BER) is a major mechanism for the repair of TMZ-induced DNA base lesions. BER inhibition, either by interrupting the delicate balance of the expression of key BER proteins or by chemical inhibitors, enhances cytotoxicity of chemotherapeutic DNA damaging agents such as TMZ. Understanding the mechanisms of enhanced cytotoxicity brought on by BER inhibition has great public health significance. By using DNA polymerase ß (Polß) deficiency as a model of BER inhibition, I report that DNA damage-induced cytotoxicity due to Polß deficiency triggers cell death dependent on PARP activation yet independent of poly(ADP-ribose) (PAR) or PAR-catabolite signaling. Cell death is rescued by the NAD+ metabolite NMN and is synergistic with inhibition of NAD+ biosynthesis demonstrating that DNA damage-induced cytotoxicity mediated via BER inhibition is primarily dependent on cellular metabolite bioavailability. I offer a mechanistic justification for the elevated alkylation-induced cytotoxicity of Polß deficient cells, suggesting a linkage between DNA repair, cell survival and cellular bioenergetics. Resistance to TMZ is partially attributed to efficient repair of TMZ-induced DNA lesions. Using the human glioma cell lines LN428 and T98G, I report here that potentiation of TMZ via BER inhibition (methoxyamine (MX), the PARP inhibitors PJ34 and ABT-888 or depletion (knockdown) of PARG) is greatly enhanced by increasing BER initiation via over-expression of MPG. I also show that MX-induced potentiation of TMZ in MPG expressing glioma cells is abrogated by elevated-expression of the rate-limiting BER enzyme Polß, suggesting that cells proficient for BER readily repair AP sites even in the presence of MX. This study demonstrates that increased initiation of BER via MPG over-expression, together with inhibition of repair following initiation, further sensitizes glioma cells to alkylating agent TMZ, suggesting that the expression level of MPG might be used to predict the effectiveness of BER inhibition-induced potentiation of TMZ in glioma cells.