Ada Elgavish

SIRT1 Is Significantly Elevated in Mouse and Human Prostate Cancer

Evidence suggests that the histone deacetylase, SIRT1, is a mediator of life span extension by calorie restriction; however, SIRT1 may paradoxically increase the risk of cancer. To better understand the relationship among SIRT1, energy balance, and cancer, two experiments were done. First, a transgenic mouse model of prostate cancer (transgenic adenocarcinoma of mouse prostate; TRAMP) was used to determine the role of energy balance on SIRT1 expression and the effect of cancer stage on SIRT1 and hypermethylated in cancer-1 (HIC-1). Second, immunohistochemistry was done on human prostate tumors to determine if SIRT1 was differentially expressed in tumor cells versus uninvolved cells. Results show that SIRT1 is not increased in the dorsolateral prostate (DLP) of calorie-restricted mice during carcinogenesis. In contrast, when examined in the DLP as a function of pathologic score, SIRT1 was significantly elevated in mice with poorly differentiated adenocarcinomas compared with those with less-advanced disease. HIC-1, which has been shown to regulate SIRT1 levels, was markedly reduced in the same tumors, suggesting that a reduction in HIC-1 may be in part responsible for the increased expression of SIRT1 in prostatic adenocarcinomas. Furthermore, immunostaining of human prostate tumors showed that cancer cells had greater SIRT1 expression than uninvolved cells. In conclusion, DLP SIRT1 expression from calorie-restricted mice was not altered during carcinogenesis. However, SIRT1 expression was increased in mice with poorly differentiated adenocarcinomas and in human prostate cancer cells. Because SIRT1 may function as a tumor promoter, these results suggest that SIRT1 should be considered as a potential therapeutic target for prostate cancer.

Osteopontin stimulates a subpopulation of quiescent human prostate epithelial cells with high proliferative potential to divide in vitro

Osteopontin (OPN) is a secreted extracellular matrix (ECM) protein found in bone, as well as associated with epithelial cells. The main objective of these studies was to test in vitro the hypothesis that interaction with OPN stimulates proliferation of a quiescent subpopulation of prostate epithelial cells with high proliferative potential.

Cancer progression in the transgenic adenocarcinoma of mouse prostate mouse is related to energy balance, body mass, and body composition, but not food intake.

Calorie restriction can inhibit or delay carcinogenesis, reportedly due to a reduction in calorie intake rather than by concurrent changes in body mass and/or composition. Our objective was to test the hypothesis that body mass and/or composition have an important effect, independent of energy intake, on the benefits or hazards associated with calorie restriction or overeating, respectively. In the first experiment, transgenic mice that spontaneously develop prostate cancer [transgenic adenocarcinoma of mouse prostate (TRAMP)] were housed at 27 degrees C or 22 degrees C and pair fed the same diet for 21 weeks (95% of ad libitum intake at 27 degrees C). In the second experiment, TRAMP mice were housed at 27 degrees C or 22 degrees C and fed the same diet ad libitum for 21 weeks. Despite a similar calorie intake, pair-fed mice at 27 degrees C (PF27) were heavier (28.3 +/- 3.3 versus 17.6 +/- 1.6 g at 21 weeks; P < 0.001; mean +/- SD) and had greater fat (6.4 +/- 2.1 versus 1.9 +/- 0.3 g; P < 0.001) and lean mass (P < 0.001) than pair-fed mice at 22 degrees C. Furthermore, PF27 mice had greater levels of serum leptin (P < 0.001), lower levels of adiponectin (P < 0.05), and a greater frequency of prostatic adenocarcinoma (P < 0.05). In contrast, ad libitum-fed mice housed at 22 degrees C consumed approximately 30% more calories than ad libitum-fed mice at 27 degrees C, but there was no difference between groups in body composition or cancer progression. These results imply that the ability of calorie restriction to inhibit or delay cancer incidence and progression is mediated in part by changes in energy balance, body mass, and/or body composition rather than calorie intake per se, suggesting that excess calorie retention, rather than consumption, confers cancer risk.

High intracellular pH in CFPAC: a pancreas cell line from a patient with cystic fibrosis is lowered by retrovirus-mediated CFTR gene transfer.

Expression of CFTR from a retroviral vector in CFPAC, a pancreatic adenocarcinoma cell line derived from a patient with Cystic Fibrosis, causes a decrease in the average intracellular pH (pHi) in these transduced clones (PLJ-CFTR), as compared to CFPAC or CFPAC transduced with control virus (PLJ clones). Whereas the average pHi calculated based on results obtained in two PLJ-CFTR clones, PLJ-CFTR-20 (n = 2) and PLJ-CFTR-6 (n = 5), was 7.46 +/- 0.07, the average pHi calculated from results obtained in CFPAC (n = 13), PLJ-6 (n = 11) and PLJ-10 (n = 3) was pH 7.83 +/- 0.11. This finding suggests that CFTR may be involved, directly or indirectly, in the regulation of pHi in the pancreas.

Polyamines stimulate d-glucose transport in isolated renal brush-border membrane vesicles

Polyamines are natural constituents of most living organisms. However, their function in normal or pathological conditions is not fully understood. We have investigated in vitro effects of polyamines on characteristic properties of isolated renal brush-border membrane vesicles in order to determine whether polyamines have a regulatory role in membrane transport processes. The polyamines putrescine, spermidine and spermine were found to stimulate D-glucose uptake. Diffusional L-glucose uptake was not altered, indicating that the polyamines affected the active transport of D-glucose, rather than inducing nonspecific changes in membrane lipid properties. The amiloride-sensitive Na+ /H + exchange was slightly inhibited by polyamines while Mg2+ -ATPase activity was stimulated. The polyamine effects could not be explained solely by the polycationic properties of these agents, since polycationic polypeptides had an opposite effect. For example, lysozyme was found to inhibit D-glucose transport. Spermine was incorporated into the trichloroacetic acid-insoluble fraction of brush-border membrane proteins. Results indicated that this incorporation process consisted of at least two components: a Ca2+ -independent component and a Ca2+ -dependent component, possibly as a result of transglutaminase activity which was present in the isolated renal brush-border membranes. By using SDS-polyacrylamide gel electrophoresis in conjunction with fluorography, [3H]spermine was shown to be incorporated into several brush-border membrane proteins, mainly the 57 kDa, 74 kDa, 100 kDa, a heavy molecular weight band (greater than 200 kDa) and a low molecular weight band (less than 10 kDa). Our results suggest that the polyamine effects on membrane function may be due to a covalent modification of membrane proteins, possibly via a transglutaminase-mediated incorporation of polyamines or to the crosslinking of membrane proteins.

Percent infarct mapping: An R1‐map‐based CE‐MRI method for determining myocardial viability distribution

Viability detection is crucial for the management of myocardial infarction (MI). Signal intensity (SI)‐based MRI methods may overestimate infarct size in vivo. In contrast to SI, the longitudinal relaxation‐rate enhancement (ΔR1) is an intrinsic parameter that is linearly proportional to the concentration of contrast agent (CA). Determining ΔR1 in the presence of an infarct‐avid persistent CA (PCA) allows determination of the per‐voxel percentage of infarcted tissue. Introduced here is a ΔR1‐based CE‐MRI method, termed percent infarct mapping (PIM), for quantifying myocardial viability following delayed PCA accumulation. In a canine MI model (N = 6), PIMs were generated using a persistent CA (PCA) and validated using triphenyltetrazolium‐chloride (TTC) histochemistry. Voxel‐by‐voxel R1 maps of the entire left ventricle (LV) were generated 24 and 48 hr after PCA administration using inversion recovery (IR) with multiple inversion times (TIs). PI values were calculated voxel by voxel. Significant correlations (P < 0.01, R = 0.97) were obtained for PI per slice (PIS) determined using PIM vs. corresponding TTC‐based values. Median deviations of PIS with PIM from that with TTC were only 1.01% and –0.53%, at 24 hr and 48 hr. Median deviations from the true infarction fraction (IF) were 1.23% and 0.49% of LV at 24 hr and 48 hr, respectively. No significant difference was found between PIM24 hr and PIM48 hr. ΔR1‐based PIM is an accurate and reproducible method for quantifying myocardial viability distribution, and thus enhances the clinical utility of CE‐MRI. Magn Reson Med, 2006.

Evidence for altered proliferative ability of progenitors of urothelial cells in interstitial cystitis.

Secondary cultures of basal urothelial cells isolated from patients with stress incontinence (7 patients), neurogenic bladder (2 patients), interstitial cystitis (IC) (27 patients), bladder rupture (1 patient) and bacterial cystitis (3 patients) grew under growth restricting conditions. All groups displayed reproducible colony size distribution, reflecting the proliferative potential distribution in the population of progenitor cells seeded. The percentage of large colonies (> 6 cells/colony), progeny of basal cells with high proliferative potential, was low in cultures from control patients with stress incontinence, neurogenic bladder or bladder rupture. Exposure of cultures from control patients with stress incontinence to lipoteichoic acid from Streptococcus faecalis, in vitro, increased the percentage of large colonies to levels statistically indistinguishable from those in untreated IC cultures. This supported the possibility that exposure of progenitors of urothelial cells to infection in vivo may cause the persistent increase in the percentage of large colonies in 80% of the IC patients tested. Given these findings, it was not surprising that the percentage of large colonies was also high in cultures from patients with acute bacterial cystitis. In conclusion, the present findings support the theoretical model for the etiology of IC we proposed based on our studies in normal urothelial cells (Elgavish et al., Journal of Cellular Physiology 169: 42-51, 52-65, 66-77, 1996): (1) The proliferative ability of a subpopulation of progenitors of urothelial cells is increased in IC; and (2) This change may be the result of recurrent exposure of progenitors of urothelial cells to injury due, possibly but not exclusively, to infection and chronic inflammation. We propose to use this change as a diagnostic tool for IC.

HIV Protease Inhibitor Ritonavir Induces Lipoatrophy in Male Mice

We investigated the effects of the HIV protease inhibitor ritonavir on body composition, serum lipids, and gene expression in C57BL/6 mice. Dual-energy X-ray absorptiometry measurements in ritonavir-treated male mice revealed whole-body lipoatrophy. In female mice fat reduction was restricted to the gonadal depot. A histopathological analysis showed no visible abnormalities in liver or adipose tissue from ritonavir-treated mice, although adipocytes were significantly smaller in diameter. Serum triglyceride levels were increased in ritonavir-treated male mice. Ritonavir was coadministered with the peroxisome proliferator-activated receptor alpha (PPARalpha) agonist gemfibrozil and the PPARgamma agonist rosiglitazone for 8 weeks. Neither drug alleviated the hypertriglyceridemia or lipoatrophy in ritonavir-treated male mice. Rather, gemfibrozil exacerbated the lipoatrophy. Ritonavir reduced basal expression of two PPARalpha target genes in liver, as well as the PPARgamma target gene phosphoenolpyruvate carboxykinase (PEPCK) in adipose tissues. Ritonavir partially inhibited induction of PPAR target genes by gemfibrozil and rosiglitazone. Gemfibrozil induced expression of fatty acid oxidation genes in liver, and this induction was less substantial when ritonavir was coadministered. Similarly, rosiglitazone induced expression of uncoupling protein-1, uncoupling protein-2, and PEPCK in adipose tissues, and this effect was partially inhibited by ritonavir. Thus, the effects of ritonavir on serum triglycerides and body composition may be due, at least in part, to an inhibition of PPAR function.

Increased sulfate uptake in skin fibroblasts isolated from cystic fibrosis patients

Sulfate uptake into skin fibroblasts from patients with cystic fibrosis is increased. Sulfate transport studies were carried out in skin fibroblasts isolated from age/sex matched cystic fibrosis and normal subjects. Sulfate transport occurred mainly via a carrier-mediated proton-stimulated S04(2)-/Cl-exchange. The capacity (Vmax) of the uptake system operating at physiological concentrations of sulfate was stimulated in cystic fibrosis, but the affinity of the carrier for sulfate was not altered.

III. Nitric oxide mediates the action of lipoteichoic acid on the function of human urothelial cells

Gram‐positive bacteria are recognized pathogens in urinary tract infections. Lipoteichoic acids, major components of the cell wall of gram‐positive bacteria, are important virulence attributes, but their mechanism of action is not well understood. We have postulated that infection‐induced altered function of progenitors of urothelial cells (UT) residing in the basal layer is likely to have long‐lasting effects on the architecture and function of the urothelium. Our earlier in vitro studies in UT of basal type, grown under growth restricting conditions, have shown that (1) treatment with lipoteichoic acid from Streptococcus faecalis (LT‐2) stimulates a subpopulation of progenitors of urothelial cells to proliferate, and (2) resulting large colonies differentiated at an increased rate under conditions simulating those in the basal layer of the urothelium. The hypothesis underlying the present studies was that nitric oxide (NO) mediated LT‐2 action on these functions of UT. Immunocytochemical studies using an antibody against inducible nitric oxide synthase (iNOS) confirmed expression of iNOS in LT‐2‐treated UT. Our hypothesis was tested by treating UT grown under growth restricting conditions (0.005% bovine pituitary extract) with LT‐2 (25 μg/ml), in the presence or absence of inhibitors of NOS (1 mM NG‐nitro‐L‐arginine methyl ester [L‐NAME]; 1 μM dexamethasone [DEXA]) or 25 μM hemoglobin, a potent inactivator of NO. Treatment with LT‐2 in the presence of these agents prevented the following effects of LT‐2 alone: (1) the stimulatory effect on proliferation of single cells, as well as within the resulting large colonies; (2) the subsequent differentiation of large colonies resulting from this proliferative activity, as indicated by distribution of β1 subunit‐containing integrins to cell‐cell contacts; (3) the inhibitory effect on the subsequent ability of LT‐2‐treated UT to attach to extracellular matrix proteins. These studies suggest that induction of NOS by LT‐2, initially aimed at restricting the replication of infectious agents, may have potential cost of damage to the host bladder by interfering with urothelial differentiation.