The role of MAPK during the activation of NK cells

Although many clinical trials investigated the use of IL-2, IL-12, and LAK in adoptive immunotherapy to treat cancer, only limited clinical success has been achieved. Better understanding of the intracellular processes that IL-2 and IL-12 utilize to generate LAK and other functions in NK cells is necessary to improve this mode of therapy. IL-2 and IL-12 stimulate extracellular signal-regulated protein kinase (ERK) and p38 MAPK in mitogen-activated T lymphocytes. The functional roles that these kinases play are still unclear. In this study, we examined whether MAPK Kinase (MKK)/ERK and/or p38 MAPK pathways are necessary for IL-2 or IL-12 to activate NK cells. We established that IL-2 activates MKK1/2/ERK pathway in freshly isolated human NK cells without any prior stimulation. Furthermore, we determined that an intact MKK/ERK pathway is necessary for IL-2 to activate NK cells to express at least four known biological responses: LAK activity, IFN-γ secretion, and CD25 and CD69 expression. Treatment of NK cells with a specific inhibitor of MKK1/2 PD98059, during the IL-2 stimulation blocked in a dose-dependent manner each of four activation parameters. Although activation of ERK was not detected in NK cells immediately after IL-12 stimulation, IL-12-induced functional activation was inhibited by the MKK1/2 inhibitor, as well. In contrast to what was observed by others in T lymphocytes, activation of p38 MAPK by IL-2 was not detected in NK cells. Additionally, a specific inhibitor of p38 MAPK (SB203850) did not inhibit IL-2-activated NK functions. These data reveal selective signaling differences between NK cells and T lymphocytes. Collectively, the data support that the MKK/ERK pathway plays a critical positive regulatory role in NK cells during activation by IL-2 and IL-12. ^

Positional candidate cloning of the RP10 form of retinitis pigmentosa

Retinitis pigmentosa (RP) is a genetically heterogeneous group of retinal degenerations that affects over one million people worldwide. To date, 11 autosomal dominant, 13 autosomal recessive, and 5 X-linked forms of retinitis pigmentosa have been identified through linkage analysis, but the disease-causing genes and mutations have been found for only half of these loci. My research uses a positional candidate cloning approach to identify the gene and mutations responsible for one type of autosomal dominant retinitis pigmentosa, RP10. The premise is that identifying the genes and mutations responsible for disease will provide insight into disease mechanisms and provide treatment options. Previous research mapped the RP10 locus to a 5cM region on chromosome 7q31 between markers D7S686 and D7S530. Linkage and fine-point haplotype analysis was used to reduce and refine the RP10 disease interval to a 4cM region located between D7S2471 and a new marker located 45,000bp telomeric of D7S461. In order to identify genes located in the RP10 interval, an extensive EST map was created of this region. Five EST clusters from this map were analyzed to determine if mutations in these genes cause the RP10 form of retinitis pigmentosa. The genomic structure of a known metabotrophic glutamate receptor, GRMS8, was determined first. DNA sequencing of GRM8 in RP10 family members did not identify any disease-causing mutations. Four other EST clusters (A170, A173, A189, and A258) were characterized and determined to be part of the same gene, UBNL1 (ubinuclein-like 1). The full-length mRNA sequence and genomic structure of UBNL1 was determined and then screened in patients. No disease-causing mutations were identified in any of the RP10 family members tested. Recent data made available with the release of the public and Celera genome assemblies indicates that UBNL1 is outside of the RP10 disease region. Despite this complication, characterization of UBNL1 is still important in the understanding of normal visual processes and it is possible that mutations in UBNL1 could cause other forms of retinopathy. The EST map and list of RP10 candidates will continue to aid others in the search for the RP10 gene and mutations. ^

Mechanisms of Ad-p53 induced apoptosis in human malignant glioma

Malignant brain tumors are one of the most challenging cancers affecting society today. In a recent survey, an estimated 17,000 annual cases were recorded with a staggering total of 13,300 deaths. A unique degree of heterogeneity typifies glial tumors and presents a challenge for solitary anti-neoplastic treatments. Tumors subsist as heterogeneous masses that progress through dysplasia to astrocytomas, mixed glioma and glioblastoma multiforme. Although traditional therapeutic approaches have provided increments of success, the median survival time remains 12 months. The urgency to improve upon current clinical protocols has encouraged alternative experimental strategies such as p53 adenoviral gene therapy (Ad-p53). This study addresses the efficacy of Ad-p53 for the treatment of glioma. Our model presents a tumor response that is unique among human cancers. Ad-p53 effectively induces apoptosis in mutant p53 expressing cells yet fails to do so in those with wildtype p53. In order to adopt Adp53 as a standard anti-cancer modality, we characterized the role of the tumor suppressor gene p53 in mediating apoptosis. We demonstrate that altering cellular p53 status through the introduction of a dominant negative mutant p53 (175H, 248W, 273H) sensitized cells to Ad-p53. We discovered that wild-type p53 expressing glioma cells retain the apoptotic machinery necessary to accomplish cell death, but have developed mechanisms that interfere with p53 signaling. Earlier studies have not addressed the mechanisms of Ad-p53 apoptosis nor the resistance exhibited by wild-type p53 glioma. To explain the divergent phenotypes, we identified apoptotic pathways activated and effectors of the response. We illustrated that modulation of the death receptor Fas/APO-1 is a principal means of Ad-p53 signaling that is impaired in wild-type p53 glioma. Moreover, the apoptotic response was found to be a multi-faceted process that engaged several caspases, most notably caspases -1, -3 and -8. Lastly, we assessed the ability of anti-apoptotic molecules Bcl-2 and CrmA to inhibit Ad-p53 apoptosis. These studies revealed that Ad-p53 is a powerful tool for inducing apoptosis that can be delayed but not inhibited by anti-apoptotic means. This work is critical for understanding the development of glioma and the phenotypic and genotypic alterations that account for tumor resistance. ^

Genetic alterations associated with prostate cancer progression

Prostate cancer is the second most commonly diagnosed cancer among men in the United States. In this study, evidence is presented to support the hypothesis that specific chromosomal aberrations (involving one or more chromosomal regions) are associated with prostate cancer progression from organ-confined to locally advanced tumors and that some aberrations seen in high frequency in metastatic tumors may also be present in a subset of primary tumors. To determine the appropriate approach to address this hypothesis, I have established a modified CGH protocol by microdissection and DOP-PCR for use in detecting chromosomal changes in clinical prostate tumor specimens that is more sensitive and accurate than conventional CGH methods. I have successfully performed the improved CGH protocol to screen for genetic changes of 24 organ confined (pT2) and 21 locally advanced (pT3b) clinical prostate cancer specimens without metastases (N0M0). Comparisons of tumors by stage or Gleason scores following contingency table analysis showed that seven regions of the genome differed significantly between pT2 and pT3b tumors or between low and high Gleason tumors suggesting that these regions may be important in local prostate cancer progression. These included losses on 6p21–25, 6q24–27, 8p, 10q25–26, 15q22–26, and 18cen–q12 as well as gain of 3p13–q13. Multivariate analyses showed that loss of 8p (step1) and loss of 6q25–26 (or 6p21–25 or 10q25–26) (step 2) were predictive of pathologic stage or Gleason groups with 80% accuracy. Additional 5–7 steps in the multivariate model increased the predictive value to 91–95%. Comparison of the CGH data from the primary prostate tumors of this study with those obtained from published literature on metastases and recurrent tumors showed that the clinically more aggressive stage pT3b tumors shared more abnormalities in high frequency with metastases and recurrent tumors than less aggressive stage pT2 tumors. Furthermore, loss of 11cen–q22 was shared only between the primary tumors and metastases while gain of Xcen–q13 and loss of 18cen–q12 were in common between primary and recurrent tumors. These analyses suggest that the multistage model of prostate cancer progression is not linear and that some early primary tumors may be predisposed to metastasize or evolve into recurrent tumors due to the presence of specific genetic alterations. ^

Arabinosylguanine: Metabolism, action, and lineage-specific cytotoxicity in human leukemia cells

9-β-D-arabinosylguanine (ara-G), an analogue of deoxyguanosine, has demonstrated T-lymphoblast selective anti-leukemia activity both in vitro and in vivo in cell lines and primary cells and in phase I investigations. The present work was initiated to identify factors that result in this selectivity. ^ The cytotoxicity of ara-G is manifest only after its phosphorylation. Experiments using cell lines transfected to overexpress specific nucleoside kinases demonstrated that the phosphorylation of ara-G to its monophosphate is by both cytoplasmic deoxycytidine kinase and mitochondria) deoxyguanosine kinase. Ara-G monophosphate is converted to its 5′-triphosphate (ara-GTP) in cells by these kinases and then incorporated into DNA. Mechanistic studies demonstrated that incorporation of ara-GTP into DNA was a necessary event for the induction of cell death. ^ Pharmacokinetic and pharmacodynamic studies utilizing three human acute leukemia cell lines, CEM (T-lymphoblastic), Raji (B-lymphoblastic), and ML-1 (myeloid) were performed. CEM cells were most sensitive to ara-G-induced inhibition of colony formation, accumulated ara-GTP at a faster rate and to a greater degree than either Raji or ML-1, but incorporated the lowest number of ara-G molecules into DNA. The position of incorporation was internal and similar in all cell lines. The terminal elimination phase of ara-GTP was >24 h and similar in these cells. Comparisons between inhibition of colony formation and ara-GTP incorporation into DNA demonstrated that while within a cell line there was correlation among these parameters, between cell lines there was no relationship between number of incorporated ara-G molecules and ara-G(TP)-mediated toxicity suggesting that there were additional factors. ^ The expression of membrane bound Fas and Fast was unchanged in all cell lines. In contrast, there was a 2-fold increase in soluble Fast, which was found exclusively in CEM cells. Ara-G-mediated apoptosis in CEM occurred from all phases of the cell cycle and was abrogated partially by Fas antagonist antibodies. These data suggest that Fas-mediated cell death due to the liberation of sFasL may be responsible for the hypersensitivity to ara-G manifested by immature T-cells such as CEM. The role of Fas in ara-G induced death of acute T-lymphoblastic leukemia cells during therapy needs to be tested. ^

Collagen I modulates growth and gene expression in prostate cancer cells

Prostate cancer is the second leading cause of male cancer-related deaths in the United States. Interestingly, prostate cancer preferentially metastasizes to skeletal tissue. Once in the bone microenvironment, advanced prostate cancer becomes highly resistant to therapeutic modalities. Several factors, such as extracellular matrix (ECM) components, have been implicated in the spread and propagation of prostatic carcinoma. In these studies, we have utilized the PC3 cell line, derived from a human bone metastasis, to investigate the influence of the predominant bone ECM protein, type I collagen, on prostate cancer cell proliferation and gene expression. We have also initiated the design and production of ribozymes to specific gene targets that may influence prostate cancer bone metastasis. ^ Our results demonstrate that PC3 cells rapidly adhere and spread on collagen I to a greater degree than on fibronectin (FN) or poly-L-lysine (PLL). Flow cytometry analysis reveals the presence of the α1, α2 and α3 collagen binding integrin subunits. The use of antibody function blocking studies reveals that PC3 cells can utilize α2β 1 and α3β1 integrins to adhere to collagen I. Once plated on collagen I, the cells exhibit increased rates of proliferation compared with cells plated on FN or tissue culture plastic. Additionally, cells plated on collagen I show increased expression of proteins associated with progression through G1 phase of the cell cycle. Inhibitor studies point to a role for phosphatidylinositol 3-kinase (PI3K), MAP kinase (MAPK), and p70 S6 kinase in collagen I-mediated PC3 cell proliferation and cyclin D1 expression. To further characterize the effect of type I collagen on prostate cancer bone metastasis, we utilized a cDNA microarray strategy to monitor type I collagen-mediated changes in gene expression. Results of this analysis revealed a gene expression profile reflecting the increased proliferation occurring on type I collagen. Microarray analysis also revealed differences in the expression of specific gene targets that may impact on prostate cancer metastasis to bone. ^ As a result of our studies on the interaction of prostate cancer cells and the skeletal ECM, we sought to develop novel molecular tools for future gene therapy of functional knockdown experiments. To this end, we developed a series of ribozymes directed against the α2 integrin and at osteopontin, a protein implicated in the metastasis of various cancers, including prostate. These ribozymes should facilitate the future study of the mechanism of prostate cancer cell proliferation, and disease progression occurring at sites of skeletal metastasis where a type I collagen-based environment predominates. ^ Together these studies demonstrate the involvement of bone ECM proteins on prostate cancer cell proliferation and suggest that they may play a significant role on the growth of prostate metastases once in the bone microenvironment. ^

A study in the molecular and cellular functions of GSK3beta in prostate adenocarcinoma

Kinases are part of a complex network of signaling pathways that enable a cell to respond to changes in environmental conditions in a regulated and coordinated way. For example, Glycogen Synthase Kinase 3 beta (GSK3β) modulates conformational changes, protein-protein interaction, protein degradation, and activation of unique domains in proteins that transduce signals from the extracellular milieu to the nucleus. ^ In this project, I investigated the expression and function that GSK3β exhibits in prostate cells. The capacity of GSK3β to regulate two transcription factors (JUN and CREB), which are known to be inversely utilized in prostate tumor cells, was measured. JUN/AP1 is constitutively activated in PC-3 cells; whereas, CREB/CRE activity is ∼20 fold less than the former. GSK3β overexpression obliterates JUN/AP1 activity. With respect to CREB GSK3β increases CREB/CRE activity. Cellular levels of active GSK3β can determine whether JUN or CREB is preferentially active in the PC-3s. Theoretically, in response to a particular cellular context or stimulus, a cell may coordinate JUN and CREB function by regulating GSK3β.^ A comparison of various prostate cell lines showed that active GSK3β is less expressed in normal prostate epithelial cells than in tumor cells. Differentially expressed active (GSK3β) may correlate with progression of prostate carcinoma. If a known marker associated with carcinoma of the prostate could be shown to be regulated by GSK3β then, further study of GSK3β may lead to a better understanding of both possible prevention of the disease and improved therapy for advanced stages. ^ The androgen receptor (AR) is an intriguing phosphoprotein whose regulation is potentially determined by a variety of kinases. One of these is (GSK3β) I found that (GSK3β) is a regulator of the androgen receptor in both the unliganded and liganded states. It can inhibit AR function as measured by reporter assays. Also, GSK3β associates with the AR at the DNA binding domain because deletion constructs expressing either the n-terminus or the c-terminus (both having the DBD in common) immunoprecipitated with GSK3β. Increased understanding of how GSK3β functions in prostate cancer would provide clues into how (1) certain signal pathways are coordinated and (2) the androgen receptor may be regulated. ^

Evaluation of candidate genes for susceptibility to spina bifida

Neural tube defects (NTDs) are the most common severely disabling birth defects in the United States, with a frequency of approximately 1–2 of every 1,000 births. This text includes the identification and evaluation of candidate susceptibility genes that confer risk for the development of neural tube defects (NTDs). The project focused on isolated meningomyelocele, also termed spina bifida (SB). ^ Spina bifida is a complex disease with multifactorial inheritance, therefore the subject population (consisting of North American Caucasians and Hispanics of Mexicali-American descent) was composed of 459 simplex SB families who were tested for genetic associations utilizing the transmission disequilibrium test (TDT), a nonparametric linkage technique. Three categories of candidate genes were studied, including (1) human equivalents of genes determined in mouse models to cause NTDs, (2) HOX and PAX genes, and (3) the MTHFR gene involved in the metabolic pathway of folate. ^ The C677T variant of the 5,10-methylenetetrahydrofolate reductase (MTHFR) gene was the first mutation in this gene to be implicated as a risk factor for NTDs. Our evaluation of the MTHFR gene provides evidence that maternal C677T homozygosity is a risk factor for upper level spina bifida defects in Hispanics [OR = 2.3, P = 0.02]. This observed risk factor is of great importance due to the high prevalence of this homozygous genotype in the Hispanic population. Additionally, maternal C677T/A1298C compound heterozygosity is a risk factor for upper level spina bifida defects in non-Hispanic whites [OR = 3.6, P = 0.03]. ^ For TDT analysis, our total population of 1128 subjects were genotyped for 54 markers from within and/or flanking the 20 candidate genes/gene regions of interest. Significant TDT findings were obtained for 3 of the 54 analyzed markers: d20s101 flanking the PAX1 gene (P = 0.019), d1s228 within the PAX7 gene (P = 0.011), and d2s110 within the PAX8 gene (P = 0.013). These results were followed-up by testing the genes directly for mutations utilizing single-strand conformational analysis (SSCA) and direct sequencing. Multiple variations were detected in each of these PAX genes; however, these variations were not passed from parent to child in phase with the positively transmitted alleles. Therefore, these variations do not contribute to the susceptibility of spina bifida, but rather are previously unreported single nucleotide polymorphisms. ^

Signal transduction of CEACAM1-L tumor suppressor

CEACAM1-L is an adhesion molecule that suppress the growth of prostate, breast, colon and endometrial tumors. In this study we defined the domain involved in CEACAM1-L tumor suppression activity. DU145 prostate cancer cells were infected with recombinant adenoviruses containing various CEACAM1-L mutant genes, and the effects of the mutant proteins on the growth of DU145 cells were assessed in a nude-mice xenograft model. We found that expression of the CEACAM1-L cytoplasm domain alone led to growth suppression of DU145 cells. These results suggest that the cytoplasmic domain of CEACAM1-L is necessary and sufficient for its growth-suppressive function. ^ The cytoplasmic domain of CEACAM1-L is presumed to be involved in a signaling pathway resulting in the suppression of tumor cell growth. It was not clear whether post-translational modification of CEACAM1-L is required for tumor suppressor function, therefore the importance of phosphorylation in growth-inhibitory signaling pathway was investigated. Full-length CEACAM1-L was found to be phosphorylated in vivo in both tyrosine and serine residues. Mutation of tyrosine 488 to phenylalanine did not abolish the tumor-suppressive activity of CEACAM1-L while mutation of serine 503 to alanine abolished the growth-inhibitory activity. In addition, mutation of serine 503 to aspartic acid produced tumor-suppressive activity similar to that of the wild-type CEACAM1-L. These results suggested that only phosphorylation at serine 503 is essential for CEACAM1-L's growth-inhibitory function in vivo. ^ Phosphorylation of CEACAM1-L may lead to its interaction with molecules in CEACAM1-L's signaling pathway. In the last part of this study we demonstrate that CEACAM1 is able to interact with the adapter protein p66Shc. p66Shc was found to be co-immunoprecipitated with full length CEACAM1-L but not with CEACAM1-L lacking its cytoplasmic tail. Additionally this interaction occurred in the absence of the tyrosine phosphorylation of CEACAM1-L. These results suggest that p66Shc is able to interact with the cytoplasmic domain of CEACAM1-L and this interaction does not require tyrosine phosphorylation. ^ In conclusion, this study suggests that CEACAM1-L signals tumor suppression through its cytoplasmic domain by initially becoming phosphorylated on serine 503. Additionally, the interaction with p66Shc may be involved in CEACAM1-L's signaling pathway. ^

Molecular alterations in nucleotide excision repair genes in malignant glioma and their relevance to malignant progression and drug resistance

Recently, it has become apparent that DNA repair mechanisms are involved in the malignant progression and resistance to therapy of gliomas. Many investigators have shown that increased levels of O6-methyl guanine DNA alkyltransferase, a DNA monoalkyl adduct repair enzyme, are correlated with resistance of malignant glioma cell lines to nitrosourea-based chemotherapy. Three important DNA excision repair genes ERCC1 (excision repair cross complementation group 1), ERCC2 (excision repair cross complementation group 2), and ERCC6 (excision repair cross complementation group 6) have been studied in human tumors. Gene copy number variation of ERCC1 and ERCC2 has been observed in primary glioma tissues. A number of reports describing a relationship between ERCC1 gene alterations and resistance to anti-cancer drugs have been also described. The levels of ERCC1 gene expression, however, have not been correlated with drug resistance in gliomas. The expression of ERCC6 gene transcribes has been shown to vary with tissue types and to be highest in the brain. There have been no comprehensive studies so far, however, of ERCC6 gene expression and molecular alterations in malignant glioma. This project examined the ERCC1 expression levels and correlated them with cisplatin resistance in malignant glioma cell lines. We also examined the molecular alterations of ERCC6 gene in primary glioma tissues and cells and analyzed whether these alterations are related to tumor progression and chemotherapy resistance. Our results indicate the presence of mutations and/or deletions in exons II and V of the ERCC6 gene, and these alterations are more frequent in exon II. Furthermore, the mutations and/or deletions in exon II were shown to be associated with increased malignant grade of gliomas. The results on the Levels of ERCC1 gene transcripts showed that expression levels correlate with cisplatin resistance. The increase in ERCC1 mRNA induced by cisplatin could be down-regulated by cyclosporin A and herbimycin A. The results of this study are likely to provide useful information for clinical treatment of human gliomas. ^

Cutaneous malignant melanoma: A study of skin cancer in the United States from 1973--1997

Cutaneous malignant melanoma (CMM) is the cancer of the melanocytes, the cells that produce the pigment melanin, and is an aggressive skin cancer that is most prevalent in the white population. Although most cases of malignant melanoma are white, black and other non-white populations also develop this disease. However, the etiologic factors involved in the development of melanoma in these lower-risk populations are not well known. Generally, survival rates of malignant melanoma have been found to be lower in blacks than for whites with similar stage of disease at diagnosis. ^ This study presents an analysis of the differences in survival between black and white cases with malignant melanoma of the skin as the only or first primary cancer, found in the National Cancer Institute Surveillance, Epidemiology and End Results (SEER) cancer registry from 1973 to 1997. A total of 54,193 cases of CMM were diagnosed in black and white patients between 1973 and 1997. Black patients tended to be older, with a mean age of 64.46 years, compared to 53.14 years for white patients. Eighty-nine percent of patients were diagnosed with CMM as the only cancer. (Abstract shortened by UMI.)^

Regulation of vascular endothelial growth factor expression in human pancreatic cancer

Pancreatic adenocarcinoma is currently the fifth-leading cause of cancer-related death in the United States. Like with other solid tumors, the growth and metastasis of pancreatic adenocarcinoma are dependent on angiogenesis. Vascular endothelial growth factor (VEGF) is a key angiogenic molecule that plays an important role in angiogenesis, growth and metastasis of many types of human cancer, including pancreatic adenocarcinoma. However, the expression and regulation of VEGF in human pancreatic cancer cells are mostly unknown. ^ To examine the hypothesis that VEGF is constitutively expressed in human pancreatic cancer cells, and can be further induced by tumor environment factors such as nitric oxide, a panel of human pancreatic cancer cell lines were studied for constitutive and inducible VEGF expression. All the cell lines examined were shown to constitutively express various levels of VEGF. To identify the mechanisms responsible for the elevated expression of VEGF, its rates of turnover and transcription were then investigated. While the half-live of VEGF was unaffected, higher transcription rates and increased VEGF promoter activity were observed in tumor cells that constitutively expressed elevated levels of VEGF. Detailed VEGF promoter analyses revealed that the region from −267 to +50, which contains five putative Sp1 binding sites, was responsible for this VEGF promoter activity. Further deletion and point mutation analyses indicated that deletion of any of the four proximal Sp1 binding sites significantly diminished VEGF promoter activity and when all four binding sites were mutated, it was completely abrogated. Consistent with these observations, high levels of constitutive Sp1 expression and DNA binding activities were detected in pancreatic cancer cells expressing high levels of VEGF. Collectively, our data indicates that constitutively expressed Sp1 leads to the constitutive expression of VEGF, and implicates that both molecules involve in the aggressive pathogenesis of human pancreatic cancer. ^ Although constitutively expressed in pancreatic cancer cells, VEGF can be further induced. In human pancreatic cancer specimens, we found that in addition to VEGF, both inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) were overexpressed, suggesting that nitric oxide might upregulate VEGF expression. Indeed, a nitric oxide donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP) significantly induced VEGF mRNA expression and protein secretion in pancreatic adenocarcinoma cells in a time- and dose-dependant manner. Using a luciferase reporter containing both the VEGF promoter and the 3′ -UTR, we showed that SNAP significantly increased luciferase activity in human pancreatic cancer cells. Notwithstanding its ability to induce VEGF in vitro, pancreatic cancer cells genetically engineered to produce NO did not exhibit increased tumor growth. This inability of NO to promote tumor growth appears to be related to NO-mediated cytotoxicity. The balance between NO mediated effects on pro-angiogenesis and cytotoxicity would determine the biological outcome of NO action on tumor cells. ^ In summary, we have demonstrated that VEGF is constitutively expressed in human pancreatic cancer cells, and that overexpression of transcription factor Sp1 is primarily responsible. Although constitutively expressed in these cells, VEGF can be further induced by NO. However, using a mouse model, we have shown that NO inhibited tumor growth by promoting cytotoxicity. These studies suggest that both Sp1 and NO may be important targets for designing potentially effective therapies of human pancreatic cancer and warrant further investigation. ^

Integrin mediated growth and apoptosis in human gastric adenocarcinoma

Integrins are important as the primary cell adhesion molecule providing information about the extracellular microenvironment to the interior of the cell to influence cellular behavior such as differentiation, proliferation and apoptosis. Apoptotic death due to loss of adhesion is termed anoikis. In this study we have obtained a parental human gastric adenocarcinoma cell line that yielded two variant lines that had differing responses to lack of adhesion. The STAD.APO cell line undergoes apoptosis when denied adherence and the STAD.ARR cell line enters into cell cycle arrest under the identical suspended conditions. We have shown that cyclin A and cyclin D mRNA and protein are down regulated when cells are denied adherence for 24 hours in tissue culture wells previously coated with poly-HEMA. To test whether cyclin A was able to rescue cells from cell cycle arrest and/or anoikis by overriding the cell cycle machinery we transfected the full length cDNA in to each cell type. Surprisingly we found that anoikis and cell cycle arrest due to suspended conditions was not affected by overexpression of cyclin A protein, but that growth under adhered conditions was reduced compared to vector alone control transfectants. Further, we transfected other cell lines; ST7, gastric cancer, MDA-MB-4.35, breast cancer, and HPB T-cell leukemic and in no case were suspended culturing conditions overcome by cyclin A. This result indicates an additional level of regulation for the cell cycle machinery. Additionally, soluble collagen was shown to be able to save from anoikis and also from cell cycle arrest while the β1 specific mAb 33B6 was only able to save from anoikis. Immunofluorescent studies show that soluble collagen creates clusters of β1 with FAK and also β1 with actin in the STAD.ARR cells but does not in the STAD.APO cells. This result indicates that the phenotypes under suspended conditions between these cell lines may diverge at their requirements for integrin ligation. Additionally we characterized the nature of anoikis by showing cytochrome c release, caspase 3, p21 and p53 activation in STAD.APO cells. Thus, our results have implications in the understanding of integrin biology and neoplastic progression. ^

An interferon-inducible protein, p202, in cancer gene therapy

Interferons (IFNs) have been shown to exert antiviral, cell growth regulatory, and immunomodulatory effects on target cells. Both type I (α and β) and type II (γ) IFNs regulate cellular activities by specifically inducing the expression or activation of endogenous proteins that perform distinct biological functions. p202 is a 52 kDa nuclear phosphoprotein known to be induced by IFNs. p202 interacts with a variety of cellular transcription and growth regulatory factors and affects their functions. ^ In this report, we showed that the expression of p202 was associated with an anti-proliferative effect on human prostate cancer cells. Cells that expressed p202 showed reduced ability to grow in soft-agar, indicating a loss of transformation phenotype. More importantly, p202 expression reduced the tumorigenicity of human prostate cancer cells. p202-expressing cells exhibit an elevated level of hypophosphorylated form of pRb, and reduced level of cyclin B1 and p55CDC. ^ Our data suggest that p202 is a growth inhibitor gene in prostate cancer cells and its expression may also suppress transformation phenotype and tumorigenicity of prostate cancer cells. ^ In addition to inhibiting in vitro cell growth, suppressing the tumorigenicity of breast cancer cells in vivo, p202 expression could sensitize breast cancer cells to apoptosis induced by TNF-α treatment. One possible mechanism contributing to this sensitization is the inactivation of NF-κB by its interaction with p202. These results provide a scientific basis for a novel therapeutic strategy that combines p202 and TNF-α treatment against breast cancer. ^ It has been reported that NF-κB is constitutively active in human pancreatic cancer cells. Since p202 interacts with NF-κB and inhibits its activity, we examined a potential p202-mediated anti-tumor activity in pancreatic cancer. We used both ectopic and orthotopic xenograft models and demonstrated that p202 expression is associated with multiple anti-tumor activities that include inhibition of tumor growth, reduced tumorigenicity, prolonged survival, and remarkably, suppression of metastasis and angiogenesis. In vitro invasion assay also showed that p202-expressing pancreatic cancer cells are less invasive than those without p202 expression. That observation was supported by the findings that p202-expressing tumors showed reduced expression of angiogenic factors such as IL-8, and VEGF by inhibiting their transcription, and p202-expressing pancreatic cancer cells have reduced level of MAP-2 activity, a secreted protease activity important for metastasis. Together, our results strongly suggest that p202 expression mediates multiple anti-tumor activities against pancreatic cancer, and that may provide a scientific basis for developing a p202-based gene therapy in pancreatic cancer treatment. ^ Importantly, we demonstrated a treatment efficacy by using p202/SN2 liposome complex in a nude mice orthotopic breast cancer, and an ectopic pancreatic cancer xenograft model, through systemic and intra-tumor injection respectively. These results suggest a feasibility of using p202/SN2 liposome in future pre-clinical gene therapy experiments. ^

Studies of the structure and function of a fibrinogen binding MSCRAMM(RTM) from Staphylococcus epidermidis

Coagulase-negative staphylococci (CNS) are recognized as important pathogens and are particularly associated with foreign body infections. S. epidermidis accounts for approximately 75% of the infections caused by CNS. Three genes, sdrF, sdrG, and sdrH, were identified by screening a S. epidermidis genomic library with a probe encompassing the serine-aspartate dipeptide repeat-encoding region (region R) of clfA from S. aureus. SdrG has significant amino acid identity to ClfA, ClfB and other surface proteins of S. aureus. SdrG is also similar to a protein (Fbe) recently described by Nilsson, et al. (Infection and Immunity, 1998, 66:2666–73) from S. epidermidis. The N-terminal domain (A region) of SdrG was expressed as a his-tag fusion protein in E. coli. In an ELISA, this protein, rSdrG(50-597) was shown to bind specifically to fibrinogen (Fg). Western ligand blot analysis showed that SdrG binds the Bβ chain of Fg. To further characterize the rSdrG(50-597)-Fg interaction, truncates of the Fg Bβ chain were made and expressed as recombinant proteins in E. coli. SdrG was shown to bind the full-length Bβ chain (1462), as well as the N-terminal three-quarters (1-341), the N-terminal one-half (1-220) and the N-terminal one-quarter (1-95) Bβ chain constructs. rSdrG(50-597) failed to bind to the recombinant truncates that lacked the N-terminal 25 amino acid residues of this polypeptide suggesting that SdrG recognizes a site within this region of the Bβ chain. Inhibition ELISAs have shown that peptide mimetics, including β1–25, and β6–20, encompassing this 25 residue region can inhibit binding of rSdrG(50-597) to Fg coated wells. Using fluorescence polarization we were able to determine an equilibrium constant (KD) for the interaction of rSdrG(50-597) with the Fg Bβ chain peptide β1–25. The labeled peptide was shown to bind to rSdrG(50-597) with a KD of 0.14 ± 0.01μM. Because rSdrG(50-597) recognizes a site in the Fg Bβ chain close to the thrombin cleavage site, we investigated the possibility of the rSdrG(50-597) site either overlapping or lying close to this cleavage site. An ELISA showed that rSdrG(50-597) binding to thrombin-treated Fg was significantly reduced. In a clot inhibition assay rSdrG(50-597) was able to inhibit fibrin clot formation in a concentration dependent manner. Furthermore, rSdrG(50-597) was able to inhibit clot formation by preventing the release of fibrinopeptide B as determined by HPLC. To further define the interaction between rSdrG(50-597) and peptide β6–20, we utilized an alanine amino acid replacement strategy. The residues in β6–20 that appear to be important in rSdrG(50-597) binding to Fg, were confirmed by the rSdrG(273-597)-β6–20 co-crystal structure that was recently solved by our collaborators at University of Alabama-Birmingham. Additionally, rSdrG(50-597) was not able to bind to Fg from different animal species, rather it bound specifically to human Fg in an ELISA. This suggests that the sequence variation between Fg Bβ chains of different species, specifically with in the N-terminal 25 residues, affects the ability of rSdrG(50-597) binding to Fg, and this may explain why S. epidermidis is primarily a human pathogen. ^