Louise Meske

A diabetes-susceptible HLA haplotype is best defined by a combination of HLA-DR and -DQ alleles.

HLA-DR4 is associated with insulin-dependent diabetes mellitus (IDDM) in many populations. Many recent studies suggest that the DR4 effect is really due to DQ3.2, an allele of the nearby DQB1 locus. We used T cell clones, MAb, and allele-specific oligonucleotides to test IDDM and control subjects for DR4 subtypes (Dw4, Dw10, Dw13, and Dw14) and for DR4-associated DQB1 alleles (DQ3.1 and DQ3.2). We find that (a) IDDM is approximately equally associated with alleles of the DRB1 locus (Dw4 and Dw10, combined relative risk, RR = 6.4) and the DQB1 locus (DQ3.2, RR = 5.9); and (b) there is significant interaction, in a statistical sense, between these DR and DQ alleles in IDDM. The only IDDM-associated DR4 haplotypes were those carrying the IDDM-associated alleles at both loci (RR = 12.1); haplotypes with Dw4 or 10 but not DQ3.2, or vice versa, had a RR less than 1. Alternative explanations include: (a) that susceptibility requires specific allelic products of both DR and DQ loci; (b) that the combination of certain DR and DQ alleles marks haplotypes with the true susceptibility allele at a third locus; or (c) that Dw4 and 10 mark haplotypes with an allele at another locus that interacts with DQ3.2. As discussed, this third locus is unlikely to be DQA1 (DQ alpha). The data thus are not easily reconciled with an exclusive effect of HLA-DQ. This information increases our ability to predict IDDM by genetic typing: in the population studied, heterozygotes DR3/[DQ3.2, Dw4] or DR3/[DQ3.2, Dw10] had a relative risk of 38.0 and an absolute risk of 1 in 15.

Plumbagin, a medicinal plant (Plumbago zeylanica)-derived 1,4-naphthoquinone, inhibits growth and metastasis of human prostate cancer PC-3M-luciferase cells in an orthotopic xenograft mouse model

We present here first time that Plumbagin (PL), a medicinal plant‐derived 1,4‐naphthoquinone, inhibits the growth and metastasis of human prostate cancer (PCa) cells in an orthotopic xenograft mouse model. In this study, human PCa PC‐3M‐luciferase cells (2 × 106) were injected into the prostate of athymic nude mice. Three days post cell implantation, mice were treated with PL (2 mg/kg body wt. i.p. five days in a week) for 8 weeks. Growth and metastasis of PC‐3M‐luciferase cells was examined weekly by bioluminescence imaging of live mice. PL‐treatment significantly (p = 0.0008) inhibited the growth of orthotopic xenograft tumors. Results demonstrated a significant inhibition of metastasis into liver (p = 0.037), but inhibition of metastasis into the lungs (p = 0.60) and lymph nodes (p = 0.27) was not observed to be significant. These results were further confirmed by histopathology of these organs. Results of histopathology demonstrated a significant inhibition of metastasis into lymph nodes (p = 0.034) and lungs (p = 0.028), and a trend to significance in liver (p = 0.075). None of the mice in the PL‐treatment group showed PCa metastasis into the liver, but these mice had small metastasis foci into the lymph nodes and lungs. However, control mice had large metastatic foci into the lymph nodes, lungs, and liver. PL‐caused inhibition of the growth and metastasis of PC‐3M cells accompanies inhibition of the expression of: 1) PKCϵ, pStat3Tyr705, and pStat3Ser727, 2) Stat3 downstream target genes (survivin and BclxL), 3) proliferative markers Ki‐67 and PCNA, 4) metastatic marker MMP9, MMP2, and uPA, and 5) angiogenesis markers CD31 and VEGF. Taken together, these results suggest that PL inhibits tumor growth and metastasis of human PCa PC3‐M‐luciferase cells, which could be used as a therapeutic agent for the prevention and treatment of human PCa.

Allelic T-cell receptor α complexes have little or no influence on susceptibility to type 1 diabetes

We performed a multiple-affected-sib study to determine if T-cell receptor alpha-chain alleles affect susceptibility to insulin-dependent diabetes mellitus. Restriction fragment length polymorphisms were used to follow the segregation of allelic T-cell receptor alpha complexes within the families. The segregation of T-cell receptor alpha alleles in 29 multiplex families revealed no significant tendency for affected sibs to share T-cell receptor alpha-chain alleles more often than would be expected by chance alone (p greater than 0.2). In contrast, the same type of analysis for HLA alleles easily detected the well-known linkage of insulin-dependent diabetes mellitus susceptibility to the HLA complex (p = 0.003). We suggest that the importance of HLA alleles in insulin-dependent diabetes mellitus susceptibility and the lack of importance of T-cell receptor alpha alleles result from the different strategies by which HLA and T-cell receptor molecules achieve antigen-binding diversity: multiple loci and allelic diversity in the case of HLA; combinatorial, junctional, and N-region diversity in the case of the T-cell receptor. In this paper we also describe three new restriction fragment length polymorphisms of the T-cell receptor alpha complex and a new method for testing the significance of linkage in multiple-affected-sib studies.

Plumbagin inhibits prostate carcinogenesis in intact and castrated PTEN knockout mice via targeting PKCε, Stat3 and epithelial to mesenchymal transition markers

Prostate cancer continues to remain the most common cancer and the second leading cause of cancer-related deaths in American males. The Pten deletions and/or mutations are frequently observed in both primary prostate cancers and metastatic prostate tissue samples. Pten deletion in prostate epithelium in mice results in prostatic intraepithelial neoplasia (PIN), followed by progression to invasive adenocarcinoma. The Pten conditional knockout mice [(Pten-loxp/loxp:PB-Cre4+) (Pten-KO)] provide a unique preclinical model to evaluate agents for efficacy for both the prevention and treatment of prostate cancer. We present here for the first time that dietary plumbagin, a medicinal plant–derived naphthoquinone (200 or 500 ppm) inhibits tumor development in intact as well as castrated Pten-KO mice. Plumbagin has shown no signs of toxicity at either of these doses. Plumbagin treatment resulted in a decrease expression of PKCϵ, AKT, Stat3, and COX2 compared with the control mice. Plumbagin treatment also inhibited the expression of vimentin and slug, the markers of epithelial-to-mesenchymal transition (EMT) in prostate tumors. In summary, the results indicate that dietary plumbagin inhibits growth of both primary and castration-resistant prostate cancer (CRPC) in Pten-KO mice, possibly via inhibition of PKCϵ, Stat3, AKT, and EMT markers (vimentin and slug), which are linked to the induction and progression of prostate cancer. Cancer Prev Res; 8(5); 375–86. ©2015 AACR.

Topically Applied Hsp90 Inhibitor 17AAG Inhibits UVR-Induced Cutaneous Squamous Cell Carcinomas

We present here that Heat shock protein 90 (Hsp90) inhibitor 17-(allylamino)-17-demethoxygeldanamycin (17AAG), when topically applied to mouse skin, inhibits ultraviolet radiation (UVR)-induced development of cutaneous squamous cell carcinoma (SCC). In these experiments, DMSO:acetone (1:40 v/v) solution of 17AAG (500nmol) was applied topically to mouse skin in conjunction with each UVR exposure (1.8 kJ/m2). The UVR source was Kodacel-filtered FS-40 sun lamps (approximately 60% UVB and 40% UVA). In independent experiments with three separate mouse lines (SKH-1 hairless mice, wild-type FVB, and PKCε overexpressing transgenic FVB mice), 17AAG treatment increased the latency and decreased both the incidence and multiplicity of UVR-induced SCC. Topical 17AAG alone or in conjunction with UVR treatments elicited neither skin nor systemic toxicity. 17AAG-caused inhibition of SCC induction was accompanied by decrease in UVR-induced: 1) hyperplasia, 2) Hsp90β-PKCε interaction, 3) expression levels of Hsp90β, Stat3, pStat3Ser727, pStat3Tyr705, pAktSer473 and matrix metalloproteinase (MMPs). The results presented here indicate that topical Hsp90 inhibitor 17AAG is effective in prevention of UVR-induced epidermal hyperplasia and SCC. One may conclude from the preclinical data presented here that topical 17AAG may be useful for prevention of UVR-induced inflammation and cutaneous SCC either developed in UVR exposed or organ transplant population.

Original ArticleTopically Applied Hsp90 Inhibitor 17AAG Inhibits UVR-Induced Cutaneous Squamous Cell Carcinomas

We present here that Heat shock protein 90 (Hsp90) inhibitor 17-(allylamino)-17-demethoxygeldanamycin (17AAG), when topically applied to mouse skin, inhibits ultraviolet radiation (UVR)-induced development of cutaneous squamous cell carcinoma (SCC). In these experiments, DMSO:acetone (1:40 v/v) solution of 17AAG (500nmol) was applied topically to mouse skin in conjunction with each UVR exposure (1.8 kJ/m2). The UVR source was Kodacel-filtered FS-40 sun lamps (approximately 60% UVB and 40% UVA). In independent experiments with three separate mouse lines (SKH-1 hairless mice, wild-type FVB, and PKCε overexpressing transgenic FVB mice), 17AAG treatment increased the latency and decreased both the incidence and multiplicity of UVR-induced SCC. Topical 17AAG alone or in conjunction with UVR treatments elicited neither skin nor systemic toxicity. 17AAG-caused inhibition of SCC induction was accompanied by decrease in UVR-induced: 1) hyperplasia, 2) Hsp90β-PKCε interaction, 3) expression levels of Hsp90β, Stat3, pStat3Ser727, pStat3Tyr705, pAktSer473 and matrix metalloproteinase (MMPs). The results presented here indicate that topical Hsp90 inhibitor 17AAG is effective in prevention of UVR-induced epidermal hyperplasia and SCC. One may conclude from the preclinical data presented here that topical 17AAG may be useful for prevention of UVR-induced inflammation and cutaneous SCC either developed in UVR exposed or organ transplant population.

Dysregulation of Autophagy Contributes to Anal Carcinogenesis.

Introduction Autophagy is an intracellular catabolic process that removes and recycles unnecessary/dysfunctional cellular components, contributing to cellular health and survival. Autophagy is a highly regulated cellular process that responds to several intracellular signals, many of which are deregulated by human papillomavirus (HPV) infection through the expression of HPV-encoded oncoproteins. This adaptive inhibitory response helps prevent viral clearance. A strong correlation remains between HPV infection and the development of squamous cell carcinoma (SCC) of the anus, particularly in HIV positive and other immunosuppressed patients. We hypothesize that autophagy is inhibited by HPV–encoded oncoproteins thereby promoting anal carcinogenesis (Fig 1). Materials and Methods HPV16 transgenic mice (K14E6/E7) and non-transgenic mice (FVB/N), both of which do not spontaneously develop anal tumors, were treated topically with the chemical carcinogen, 7,12-Dimethylbenz[a]anthracene (DMBA), to induce anal cancer. The anuses at different time points of treatment (5, 10, 15 and 20 weeks) were analyzed using immunofluorescence (IF) for two key autophagy marker proteins (LC3β and p62) in addition to histological grading. The anuses from the K14E6/E7 mice were also analyzed for visual evidence of autophagic activity by electron microscopy (EM). To see if there was a correlation to humans, archival anal specimens were assessed histologically for grade of dysplasia and then analyzed for LC3β and p62 protein content. To more directly examine the effect of autophagic inhibition on anal carcinogenesis, nontransgenic mice that do not develop anal cancer with DMBA treatment were treated with a known pharmacologic inhibitor of autophagy, chloroquine, and examined for tumor development and analyzed by IF for autophagic proteins. Results Histologically, we observed the progression of normal anoderm to invasive SCC with DMBA treatment in K14E6/E7 mice but not in nontransgenic, syngeneic FVB/N background control mice. With the development of low-grade dysplasia in the K14E6/E7 mice, there was an increase in both punctate LC3β and p62 expression while EM revealed increased autophagosomes without evidence of autophagolysosomes. These observations are consistent with autophagy being inhibited at a later stage in the autophagic process. In contrast, in high-grade dysplasia and SCC in the DMBA-treated K14E6/E7 mice, there were decreased levels of p62 with a continued increase in punctate LC3β expression by IF, while autophagolysosomes were seen on EM, consistent with the process of autophagy proceeded to completion. Similar findings, including histological grade dependent changes in LC3β and p62 expression, were noted with human samples upon analysis of IF. Finally, with pharmacologic inhibition of autophagy in DMBA-treated, nontrangenic FVB/N mice, there was a significant increase in anal cancer development similar to that observed in DMBA- treated K14E6/E7 mice. Conclusion Autophagic dysregulation is noted early on in HPV-associated anal carcinogenesis (low-grade dysplasia), with normalization of the autophagic process arising in late stages of HPV-associated anal carcinogenesis (high-grade dysplasia and invasive carcinoma).

Cholecystokinin expression in the β-cell leads to increased β-cell area in aged mice and protects from streptozotocin-induced diabetes and apoptosis

Cholecystokinin (CCK) is a peptide hormone produced in the gut and brain with beneficial effects on digestion, satiety, and insulin secretion. CCK is also expressed in pancreatic β-cells, but only in models of obesity and insulin resistance. Whole body deletion of CCK in obese mice leads to reduced β-cell mass expansion and increased apoptosis. We hypothesized that islet-derived CCK is important in protection from β-cell apoptosis. To determine the specific role of β-cell-derived CCK in β-cell mass dynamics, we generated a transgenic mouse that expresses CCK in the β-cell in the lean state (MIP-CCK). Although this transgene contains the human growth hormone minigene, we saw no expression of human growth hormone protein in transgenic islets. We examined the ability of MIP-CCK mice to maintain β-cell mass when subjected to apoptotic stress, with advanced age, and after streptozotocin treatment. Aged MIP-CCK mice have increased β-cell area. MIP-CCK mice are resistant to streptozotocin-induced diabetes and exhibit reduced β-cell apoptosis. Directed CCK overexpression in cultured β-cells also protects from cytokine-induced apoptosis. We have identified an important new paracrine/autocrine effect of CCK in protection of β-cells from apoptotic stress. Understanding the role of β-cell CCK adds to the emerging knowledge of classic gut peptides in intraislet signaling. CCK receptor agonists are being investigated as therapeutics for obesity and diabetes. While these agonists clearly have beneficial effects on body weight and insulin sensitivity in peripheral tissues, they may also directly protect β-cells from apoptosis.

The role of pharmacologic modulation of autophagy on anal cancer development in an HPV mouse model of carcinogenesis

Autophagy is an intracellular, catabolic process that maintains cellular health. We examined the response of pharmacologic modulation of autophagy in an HPV mouse model of anal carcinogenesis. K14E6/E7 mice were treated with the topical carcinogen DMBA weekly and assessed for tumors over 20 weeks. Concurrently, they were given either chloroquine or BEZ235, to inhibit or induce autophagy, respectively. Time to tumor onset was examined. Immunofluorescence (IF) was performed for LC3β and p62 to examine autophagy. All DMBA treated K14E6/E7 mice developed anal cancer, contrary to zero of the no DMBA treated mice. Chloroquine plus DMBA resulted in a significant decrease in the time to tumor onset compared to K14E6/E7 treated with DMBA. Only 40% BEZ235 plus DMBA treated mice developed anal cancer. Autophagic induction with DMBA and BEZ235, and autophagic inhibition with chloroquine were confirmed via IF. Anal carcinogenesis can be inhibited or induced via pharmacologic modulation of autophagy.

Genetic inhibition of autophagy in a transgenic mouse model of anal cancer

Background: The dynamic role of autophagy in cancer development is a topic of considerable research and debate. Previously published studies have shown that anal cancer development can be promoted or prevented with the pharmacologic inhibition or induction, respectively, of autophagy in a human papillomavirus (HPV) mouse model. However, these results are confounded by the fact that the drugs utilized are known to affect other pathways besides autophagy. It has also been shown that autophagic inhibition occurs in the setting of HPV16 oncoprotein expression (E6 and E7) and correlates with increased susceptibility to anal carcinogenesis. Materials and Methods: In this study, we employed a conditional, genetic, autophagic (Atg7) knockout mouse model to determine conclusively that autophagy has a role in anal cancer development, in the absence or presence of E6 and E7. Results: In mice lacking both HPV16 oncogenes, knockout of autophagy followed by exposure to a carcinogen resulted in a tumor incidence of 40%, compared to 0% in mice treated with a carcinogen alone with an intact autophagic pathway (P = 0.007). In mice expressing either one or both HPV16 oncoproteins, the addition of genetic knockout of autophagy to carcinogen treatment did not lead to a significant difference in tumor incidence compared to carcinogen treatment alone, consistent with the ability of HPV oncogenes to inhibit autophagy in themselves. Conclusions: These results provide the first conclusive evidence for the distinct role of autophagy in anal carcinogenesis, and suggest that autophagy is a plausible target for therapies aimed at reducing anal dysplasia and anal cancer development.