Jai Parkash

Calcium wave signaling in cancer cells

Ca(2+) functions as an important signaling messenger right from beginning of life to the final moments of the end of life. Ca(2+) is needed at several steps of the cell cycle such as early G(1), at the G(1)/S, and G(2)/M transitions. The Ca(2+) signals in the form of time-dependent changes in intracellular Ca(2+) concentrations, [Ca(2+)](i), are presented as brief spikes organized into regenerative Ca(2+) waves. Ca(2+)-mediated signaling pathways have also been shown to play important roles in carcinogenesis such as transformation of normal cells to cancerous cells, tumor formation and growth, invasion, angiogenesis and metastasis. Since the global Ca(2+) oscillations arise from Ca(2+) waves initiated locally, it results in stochastic oscillations because although each cell has many IP(3)Rs and Ca(2+) ions, the law of large numbers does not apply to the initiating event which is restricted to very few IP(3)Rs due to steep Ca(2+) concentration gradients. The specific Ca(2+) signaling information is likely to be encoded in a calcium code as the amplitude, duration, frequency, waveform or timing of Ca(2+) oscillations and decoded again at a later stage. Since Ca(2+) channels or pumps involved in regulating Ca(2+) signaling pathways show altered expression in cancer, one can target these Ca(2+) channels and pumps as therapeutic options to decrease proliferation of cancer cells and to promote their apoptosis. These studies can provide novel insights into alterations in Ca(2+) wave patterns in carcinogenesis and lead to the development of newer technologies based on Ca(2+) waves for the diagnosis and therapy of cancer.

Reactive Oxygen Species via Redox Signaling to PI3K/AKT Pathway Contribute to the Malignant Growth of 4-Hydroxy Estradiol-Transformed Mammary Epithelial Cells

The purpose of this study was to investigate the effects of 17-β-estradiol (E2)-induced reactive oxygen species (ROS) on the induction of mammary tumorigenesis. We found that ROS-induced by repeated exposures to 4-hydroxy-estradiol (4-OH-E2), a predominant catechol metabolite of E2, caused transformation of normal human mammary epithelial MCF-10A cells with malignant growth in nude mice. This was evident from inhibition of estrogen-induced breast tumor formation in the xenograft model by both overexpression of catalase as well as by co-treatment with Ebselen. To understand how 4-OH-E2 induces this malignant phenotype through ROS, we investigated the effects of 4-OH-E2 on redox-sensitive signal transduction pathways. During the malignant transformation process we observed that 4-OH-E2 treatment increased AKT phosphorylation through PI3K activation. The PI3K-mediated phosphorylation of AKT in 4-OH-E2-treated cells was inhibited by ROS modifiers as well as by silencing of AKT expression. RNA interference of AKT markedly inhibited 4-OH-E2-induced in vitro tumor formation. The expression of cell cycle genes, cdc2, PRC1 and PCNA and one of transcription factors that control the expression of these genes – nuclear respiratory factor-1 (NRF-1) was significantly up-regulated during the 4-OH-E2-mediated malignant transformation process. The increased expression of these genes was inhibited by ROS modifiers as well as by silencing of AKT expression. These results indicate that 4-OH-E2-induced cell transformation may be mediated, in part, through redox-sensitive AKT signal transduction pathways by up-regulating the expression of cell cycle genes cdc2, PRC1 and PCNA, and the transcription factor – NRF-1. In summary, our study has demonstrated that: (i) 4-OH-E2 is one of the main estrogen metabolites that induce mammary tumorigenesis and (ii) ROS-mediated signaling leading to the activation of PI3K/AKT pathway plays an important role in the generation of 4-OH-E2-induced malignant phenotype of breast epithelial cells. In conclusion, ROS are important signaling molecules in the development of estrogen-induced malignant breast lesions.

Long Work Hours Is Associated With Suboptimal Glycemic Control Among US Workers With Diabetes

BACKGROUND Increasing numbers of US workers are diabetic. We assessed the relationship between glycemic control and work hours and type of occupation among employed US adults with type 2 diabetes. METHODS Data were obtained from the 1999-2004 National Health and Nutrition Examination Survey (NHANES). A representative sample of employed US adults ≥20 years with self-reported type 2 diabetes (n = 369) was used. Two dichotomous glycemic control indicators, based on various HbA1c level cut-points, were used as dependent variables in weighted logistic regression analyses with adjustment for confounders. RESULTS Adults working over 40 hr/week were more likely to have suboptimal glycemic control (HbA1c ≥ 7%) compared to those working 20 hr or less (odds ratio = 5.09; 95% confidence interval: [1.38-18.76]). CONCLUSIONS Work-related factors, such as number of hours worked, may affect the ability of adults with type 2 diabetes to reach and maintain glycemic control goals. These factors should be considered in the development of workplace policies and accommodations for the increasing number of workers with type 2 diabetes.

L-histidine sensing by calcium sensing receptor inhibits voltage-dependent calcium channel activity and insulin secretion in β-cells

AIMS Our goal was to test the hypothesis that the histidine-induced activation of calcium sensing receptor (CaR) can regulate calcium channel activity of L-type voltage dependent calcium channel (VDCC) due to increased spatial interaction between CaR and VDCC in β-cells and thus modulate glucose-induced insulin secretion. MAIN METHODS Rat insulinoma (RINr1046-38) insulin-producing β-cells were cultured in RPMI-1640 medium on 25 mm diameter glass coverslips in six-well culture plates in a 5% CO(2) incubator at 37°C. The intracellular calcium concentration, [Ca(2+)](i), was determined by ratio fluorescence microscopy using Fura-2AM. The spatial interactions between CaR and L-type VDCC in β-cells were measured by immunofluorescence confocal microscopy using a Nikon C1 laser scanning confocal microscope. The insulin release was determined by enzyme-linked immunosorbent assay (ELISA). KEY FINDINGS The addition of increasing concentrations of L-histidine along with 10 mM glucose resulted in 57% decrease in [Ca(2+)](i). The confocal fluorescence imaging data showed 5.59 to 8.62-fold increase in colocalization correlation coefficient between CaR and VDCC in β-cells exposed to L-histidine thereby indicating increased membrane delimited spatial interactions between these two membrane proteins. The insulin ELISA data showed 54% decrease in the 1st phase of glucose-induced insulin secretion in β-cells exposed to increasing concentrations of L-histidine. SIGNIFICANCE L-histidine-induced increased spatial interaction of CaR with VDCC can inhibit calcium channel activity of VDCC and consequently regulate glucose-induced insulin secretion by β-cells. The L-type VDCC could therefore be a potential therapeutic target in diabetes.

Glucose-mediated spatial interactions of voltage dependent calcium channels and calcium sensing receptor in insulin producing β-cells.

AIMS The voltage dependent calcium channel (VDCC) e.g., L-type VDCC plays critical roles in the spatio-temporal regulation of intracellular calcium concentration ([Ca(2+)](i)) and insulin secretion by β-cell. This study describes the involvement of 2.5 to 15mM glucose-induced spatial interactions between a calcium sensing receptor (CaR) and L-type VDCC in controlling Ca(2+) channel activity and insulin secretion in β-cells in association with the nuclear translocation of a transcription factor nuclear factor kappa B (NF-κB). MAIN METHODS The insulin producing β-cells were exposed to 2.5, 5, 7.5, 10, and 15 mM glucose for 24 h at 37 °C. The confocal fluorescence imaging data was obtained by using antibodies against CaR and L-type VDCC. The nuclear translocation of NF-κB was measured by confocal fluorescence imaging using antibody against NF-κB. The insulin release was determined by enzyme-linked immunosorbent assay (ELISA). KEY FINDINGS The confocal imaging data showed 6 to 12-fold enhancement in the colocalization correlation coefficient between CaR and VDCC in β-cells exposed to glucose thereby indicating increased membrane delimited spatial interactions between these two membrane proteins. The confocal fluorescence imaging data showed that addition of glucose to β-cells led to 1.8 to 2.7-fold increase in the nuclear translocation of NF-κB. The insulin ELISA data showed a significant increase in the 1st phase of glucose-induced insulin secretion in β-cells exposed to increasing concentrations of glucose. SIGNIFICANCE The results described in the present study further strengthen that VDCC and CaR can interact spatially to allow control over calcium channel activity and therefore glucose-induced insulin secretion by β-cells.

The Presence of Human Chorionic Gonadotropin/Luteinizing Hormone Receptors in Pancreatic β-Cells

We investigated the possible presence of functional human chorionic gonadotropin (hCG)/luteinizing hormone (LH) receptors in β-cells of pancreas, using a combination of techniques on hCG/LH receptor knockout mice, immortalized rat insulinoma cells, and human pancreatic islets. The results showed the presence of receptors and their activation resulted in a dose-dependent increase in glucose-induced release of insulin. These findings place hCG and LH among the regulators of insulin release with potential implications for insulin-level changes during the periods of altered hCG and LH secretion.

Strict glycemic control and mortality risk among US adults with type 2 diabetes

OBJECTIVE To assess the relationships between strict HbA1c levels and mortality risk among adults with type 2 diabetes by age, insulin therapy, and hypertension comorbidity. METHODS Data of adult participants with type 2 diabetes from the third National Health and Nutrition Examination Survey (1988-1994) and its linked mortality file (with follow-up death up to 2000) were used. RESULTS Having strict glycemic control (i.e., HbA1c ≤6.5%) was associated with a lower risk of mortality (hazards ratio=0.69; 95% confidence interval=0.48-0.98). However, among those with strict glycemic control levels, statistically significant results were not found. CONCLUSION Reaching strict glycemic control levels in the general US population with type 2 diabetes appears to be associated with lower mortality. Further research is needed as to how strict glycemic control affects certain diabetic groups.

Calcium Oscillations and Waves in Cells

From beginning of the life to final moment of the life, Ca(2+) functions as an important signaling messenger. The intracellular Ca(2+) concentration, [Ca(2+)](i), in resting cells is normally maintained at around 100 nM with a very steep ∼20,000 times concentration gradient of Ca(2+) between extracellular and intracellular compartments. Ca(2+) signals in the form of time-dependent changes in [Ca(2+)](i) appear as brief spikes that are organized into regenerative Ca(2+) waves. The release of Ca(2+) from internal stores plays a key role in regulating such Ca(2+) signals. Since global Ca(2+) oscillations arise from Ca(2+) waves initiated locally, it results in generation of stochastic Ca(2+) oscillations. In addition, the hierarchical organization of signaling structures translate the molecular fluctuations of single channels to the whole cell leading to formation of stochastic media. Several recent observations indicate that [Ca(2+)](i) changes are fluctuation driven as opposed to a typical deterministic intracellular reaction-diffusion system model. Elucidation of this signaling mechanism can provide detailed knowledge of relationship between cell signaling and cell physiology of living systems.

Tumor necrosis factor alpha inducing spatial interactions between calcium-sensing receptor and L-type voltage-dependent calcium channel.

The temporal and spatial regulation of intracellular calcium concentration ([Ca2+]i) is very crucial for maintaining calcium ion homeostasis within cells, and consequently in the regulation of cellular functions such as β cell replication and differentiation, insulin secretion, and apoptosis. Calcium ion regulatory proteins playing major roles in these processes include L‐type voltage‐dependent calcium channels (L‐type VDCCs) and calcium‐sensing receptors (CaRs). Tumor necrosis factor alpha (TNF‐α), a cytokine, is widely known to activate nuclear factor‐kappa B (NF‐κB) transcription in β cells. Confocal fluorescence imaging data suggest increased co‐localization of CaRs with L‐type VDCCs upon treatment of β cells with TNF‐α, thereby indicating increased membrane‐delimited spatial interactions between these two membrane proteins.

The growing cohort of seniors with HIV/AIDS: changing the scope of Medicare Part D.

Abstract HIV/AIDS medications are generally expensive and government assistance is often necessary to limit high out-of-pocket patient costs. Lowered patient out-of-pocket costs were the objective of government involvement in drug provision through legislation creating Medicare Part D. However, the Medicare program faces a surge in those beneficiaries living longer on more effective antiretroviral drugs. Higher prevalence of HIV/AIDS patients means more opportunity for transmission of the infection and recidivistic behavior such as non-adherence to medication regimens. Along with the resulting increased frequency of opportunistic infections in HIV/AIDS patients comes the requirement for aggressive pharmacological treatment. To meet this need, Medicare Part D provides drugs for the treatment of opportunistic infections occurring in HIV/AIDS patients. Problematically, though, Medicare Part D contains so many choices that it tends to overwhelm patients and sometimes even the providers and insurance companies as well. The multiplicity of choices in this highly complex program for the aged and infirm often leads to confusion and incorrect choices by beneficiaries. Furthermore, the advent of tiered cost-sharing or formulary management by Medicare Part D providers, besides controlling out-of-pocket costs, controls which medications are covered and limits the quantity that is dispensed. HIV/AIDS treatment in the present day requires a highly accessible medication provision program that is only now beginning to evolve as Medicare Part D.