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Objective: The presence of mild thiamine deficiency and prevention of micro-vascular complications by thiamine repletion in experimental diabetes has raised concern over thiamine status in clinical diabetes. We recently found plasma thiamine deficiency was prevalent in type 1 and type 2 diabetic patients in the United Kingdom. The aim of this study is to investigate the expression of two membrane transport proteins in red blood cells (RBCs) and mononuclear cells (MNLs): human thiamine transporter (hTHTR)-1 - a transporter of thiamine, and the reduced-folate carrier-1 (RFC-1) - a transporter of thiamine monophosphate. Methods: Diabetic patients (Type 1 diabetic patients n=12; Type 2 diabetic patients n=12) were recruited from those attending the Diabetes clinic at Colchester General Hospital; and normal healthy (n=10) subjects from partners and friends of the patients and investigators. Protein expression of the thiamine transporters was determined by Western-blot analysis. Results: Western blotting of RBC and MNL membrane proteins from diabetic patients showed increased contents of these transporters compared to the normal subjects. Thiamine transporter/β-actin blot intensity ratios (median, minimum - maximum) were: RBCs - controls (n = 10) - THTR - 10.69 (0.12 - 1.43) and RFC-1 0.042 (0.012 - 0.093); type 1 diabetes In = 12) - THTR-1 1.06 (0.64 - 2.02), + 54%, P<0.05 and RFC-1 0.146 (0.021 - 0.902), + 2-fold%, P<0.01; and type 2 diabetes (n = 12) - THTR- 1 1.20 (0.62 - 2.39), +74%, P<0.01 and RFC-1 0.206 (0.038 - 1.382), + 4-fold, P<0.001. MNLs - controls (n = 10) - THTR-1 0.71 (0.16 - 1.58) and RFC-1 1.02 (0.41 - 2.26); type 1 diabetes (n = 12) - THTR-1 3.78 (1.65 - 8.33), + 5-fcld, P<0.001 and RFC-1 4.11 (0.49 - 9.15), + 4-fold, P<0.01; and type 2 diabetes (n = 12) - THTR-1 1.45 (0.54 - 8.26), + 2-fold, P<0.01 and RFC-1 1.77 (0.49 - 9.73), +74%, P>0.05 (Mann Whitney-U). Conclusion: This study indicates both type 1 and type 2 diabetic patients exhibit increased contents of thiamine transporters in RBCs (THTR-1 and RFC-1) and MNLs (THTR-1 only) compared to normal subjects. Increased thiamine transporter express ion may be protective response to plasma thiamine deficiency. [ABSTRACT FROM AUTHOR]

Lymphangioleiomyomatosis (LAM) is a female-specific cystic lung disease in which tuberous sclerosis complex 2 (TSC2)-deficient LAM cells, LAM-associated fibroblasts (LAFs), and other cell types infiltrate the lungs. LAM lesions can be associated with type II alveolar epithelial (AT2) cells. We hypothesized that the behavior of AT2 cells in LAM is influenced locally by LAFs. We tested this hypothesis in the patient samples and in vitro. In human LAM lung, nodular AT2 cells show enhanced proliferation when compared with parenchymal AT2 cells, demonstrated by increased Ki67 expression. Furthermore, nodular AT2 cells express proteins associated with epithelial activation in other disease states including matrix metalloproteinase 7, and fibroblast growth factor 7 (FGF7). In vitro, LAF-conditioned medium is mitogenic and positively chemotactic for epithelial cells, increases the rate of epithelial repair, and protects against apoptosis. In vitro, LAM patient-derived TSC2 null cells cocultured with LAFs upregulate LAF expression of the epithelial chemokine and mitogen FGF7, a potential mediator of fibroblast-epithelial cross talk, in a mechanistic target of rapamycin (mTOR)-dependent manner. In a novel in vitro model of LAM, ex vivo cultured LAM lung-derived microtissues promote both epithelial migration and adhesion. Our findings suggest that AT2 cells in LAM display a proliferative, activated phenotype and fibroblast accumulation following LAM cell infiltration into the parenchyma contributes to this change in AT2 cell behavior. Fibroblast-derived FGF7 may contribute to the cross talk between LAFs and hyperplastic epithelium in vivo, but does not appear to be the main driver of the effects of LAFs on epithelial cells in vitro.

Colorectal cancer (CRC) is one of the top three cancer-related causes of death worldwide. FBXW7 is a known tumor-suppressor gene, commonly mutated in CRC and in a variety of other epithelial tumors. Low expression of FBXW7 is also associated with poor prognosis. Loss of FBXW7 sensitizes cancer cells to certain drugs, while making them more resistant to other types of chemotherapies. However, is not fully understood how epithelial cells within normal gut and primary tumors respond to potential cancer therapeutics. We have studied genetically engineered mice in which the fbxw7 gene is conditionally knocked-out in the intestine (fbxw7(∆G)). To further investigate the mechanism of Fbxw7-action, we grew intestinal crypts from floxed-fbxw7 (fbxw7(fl/fl)) and fbxw7(ΔG) mice, in a Matrigel-based organoid (mini-gut) culture. The fbxw7(ΔG) organoids exhibited rapid budding events in the crypt region. Furthermore, to test organoids for drug response, we exposed day 3 intestinal organoids from fbxw7(fl/fl) and fbxw7(∆G) mice, to various concentrations of 5-fluorouracil (5-FU) for 72 hours. 5-FU triggers phenotypic differences in organoids including changing shape, survival, resistance, and death. 5-FU however, rescues the drug-resistance phenotype of fbxw7(ΔG) through the induction of terminal differentiation. Our results support the hypothesis that a differentiating therapy successfully targets FBXW7-mutated CRC cells.

The Fbxw7 (F-box/WD repeat-containing protein 7; also called CDC4, Sel10, Ago, and Fbw7) component of the SCF (Skp1/Cullin/F-box protein) E3 ubiquitin ligase complex acts as a tumor suppressor in several tissues and targets multiple transcriptional activators and protooncogenes for ubiquitin-mediated degradation. To understand Fbxw7 function in the murine intestine, in this study, we specifically deleted Fbxw7 in the murine gut using Villin-Cre (Fbxw7(ΔG)). In wild-type mice, loss of Fbxw7 in the gut altered homeostasis of the intestinal epithelium, resulted in elevated Notch and c-Jun expression, and induced development of adenomas at 9-10 mo of age. In the context of APC (adenomatous polyposis coli) deficiency (Apc(Min/+) mice), loss of Fbxw7 accelerated intestinal tumorigenesis and death and promoted accumulation of β-catenin in adenomas at late but not early time points. At early time points, Fbxw7 mutant tumors showed accumulation of the DEK protooncogene. DEK expression promoted cell division and altered splicing of tropomyosin (TPM) RNA, which may also influence cell proliferation. DEK accumulation and altered TPM RNA splicing were also detected in FBXW7 mutant human colorectal tumor tissues. Given their reduced lifespan and increased incidence of intestinal tumors, Apc(Min/+)Fbxw7(ΔG) mice may be used for testing carcinogenicity and drug screening.

Interactions between transcription and signaling are fundamentally important for understanding both the structure and function of genetic pathways and their role in diseases such as cancer. The finding that beta-catenin/TCF4 and JNK/c-Jun cooperate has important implications in carcinogenesis. Previously, we found that binding of c-Jun and beta-catenin/TCF4 to the c-jun promoter is dependent upon JNK activity, thus one role for this complex is to contribute to the repression and/or activation of genes that may mediate cell maintenance, proliferation, differentiation, and death, whereas deregulation of these signals may contribute to carcinogenesis. Here we address the functional links reported between activated beta-catenin/JNK signaling pathways, their component genes, and their common targets, and discuss how alterations in the properties of these genes lead to the development of cancer.