MECHANISM-BASED STRATEGIES TO ENHANCE THE ACTIONS OF A

Heat shock protein 90 (HSP90) is an abundant molecular chaperone that regulates the functional stability of client oncoproteins, such as STAT3, Raf-1 and Akt, which play a role in the survival of malignant cells. The chaperone function of HSP90 is driven by the binding and hydrolysis of ATP. The geldanamycin analog, 17-AAG, binds to the ATP pocket of HSP90 leading to the degradation of client proteins. However, treatment with 17-AAG results in the elevation of the levels of antiapoptotic proteins HSP70 and HSP27, which may lead to cell death resistance. The increase in HSP70 and HSP27 protein levels is due to the activation of the transcription factor HSF-1 binding to the promoter region of HSP70 and HSP27 genes. HSF-1 binding subsequently promotes HSP70 and HSP27 gene expression. Based on this, I hypothesized that inhibition of transcription/translation of HSP or client proteins would enhance 17-AAG-mediated cytotoxicity. Multiple myeloma (MM) cell lines MM.1S, RPMI-8226, and U266 were used as a model. To test this hypothesis, two different strategies were used. For the first approach, a transcription inhibitor was combined with 17-AAG. The established transcription inhibitor Actinomycin D (Act D), used in the clinic, intercalates into DNA and blocks RNA elongation. Stress inducible (HSP90á, HSP70 and HSP27) and constitutive (HSP90â and HSC70) mRNA and protein levels were measured using real time RT-PCR and immunoblot assays. Treatment with 0.5 µM 17-AAG for 8 hours resulted in the induction of all HSP transcript and protein levels in the MM cell lines. This induction of HSP mRNA levels was diminished by 0.05 µg/mL Act D for 12 hours in the combination treatment, except for HSP70. At the protein level, Act D abrogated the 17-AAG-mediated induction of all HSP expression levels, including HSP70. Cytotoxic evaluation (Annexin V/7-AAD assay) of Act D in combination with 17-AAG suggested additive or more than additive interactions. For the second strategy, an agent that affected bioenergy production in addition to targeting transcription and translation was used. Since ATP is necessary for the proper folding and maturation of client proteins by HSP90, ATP depletion should lead to a decrease in client protein levels. The transcription and translation inhibitor 8-Chloro-Adenosine (8-Cl-Ado), currently in clinical trials, is metabolized into its cytotoxic form 8-Cl-ATP causing a parallel decrease of the cellular ATP pool. Treatment with 0.5 µM 17-AAG for 8 hours resulted in the induction of all HSP transcript and protein levels in the three MM cell lines evaluated. In the combination treatment, 10 µM 8-Cl-Ado for 20 hours did not abrogate the induction of HSP mRNA or protein levels. Since cellular bioenergy is necessary for the stabilization of oncoproteins by HSP90, immunoblot assays analyzing for expression levels of client proteins such as STAT3, Raf-1, and Akt were performed. Immunoblot assays detecting for the phosphorylation status of the translation repressor 4E-BP1, whose activity is modulated by upstream kinases sensitive to changes in ATP levels, were also performed. The hypophosphorylated state of 4E-BP1 leads to translation repression. Data indicated that treatment with 17-AAG alone resulted in a minor (<10%) change in STAT3, Raf-1, and Akt protein levels, while no change was observed for 4E-BP1. The combination treatment resulted in more than 50% decrease of the client protein levels and hypophosphorylation of 4E-BP1 in all MM cell lines. Treatment with 8-Cl-Ado alone resulted in less than 30% decrease in client protein levels as well as a decrease in 4E-BP1 phosphorylation. Cytotoxic evaluation of 8-Cl-Ado in combination with 17-AAG resulted in more than additive cytotoxicity when drugs were combined in a sequential manner. In summary, these data suggest that the mechanism-based combination of agents that target transcription, translation, or decrease cellular bioenergy with 17-AAG results in increase cytotoxicity when compared to the single agents. Such combination strategies may be applied in the clinic since these drugs are established chemotherapeutic agents or currently in clinical trials.

Excess stroke in Mexican Americans compared with non-Hispanic Whites: the Brain Attack Surveillance in Corpus Christi Project.

Mexican Americans are the largest subgroup of Hispanics, the largest minority population in the United States. Stroke is the leading cause of disability and third leading cause of death. The authors compared stroke incidence among Mexican Americans and non-Hispanic Whites in a population-based study. Stroke cases were ascertained in Nueces County, Texas, utilizing concomitant active and passive surveillance. Cases were validated on the basis of source documentation by board-certified neurologists masked to subjects' ethnicity. From January 2000 to December 2002, 2,350 cerebrovascular events occurred. Of the completed strokes, 53% were in Mexican Americans. The crude cumulative incidence was 168/10,000 in Mexican Americans and 136/10,000 in non-Hispanic Whites. Mexican Americans had a higher cumulative incidence for ischemic stroke (ages 45-59 years: risk ratio = 2.04, 95% confidence interval: 1.55, 2.69; ages 60-74 years: risk ratio = 1.58, 95% confidence interval: 1.31, 1.91; ages >or=75 years: risk ratio = 1.12, 95% confidence interval: 0.94, 1.32). Intracerebral hemorrhage was more common in Mexican Americans (age-adjusted risk ratio = 1.63, 95% confidence interval: 1.24, 2.16). The subarachnoid hemorrhage age-adjusted risk ratio was 1.57 (95% confidence interval: 0.86, 2.89). Mexican Americans experience a substantially greater ischemic stroke and intracerebral hemorrhage incidence compared with non-Hispanic Whites. As the Mexican-American population grows and ages, measures to target this population for stroke prevention are critical.

Progenitor cell therapies for traumatic brain injury: barriers and opportunities in translation.

Traumatic brain injury (TBI) directly affects nearly 1.5 million new patients per year in the USA, adding to the almost 6 million cases in patients who are permanently affected by the irreversible physical, cognitive and psychosocial deficits from a prior injury. Adult stem cell therapy has shown preliminary promise as an option for treatment, much of which is limited currently to supportive care. Preclinical research focused on cell therapy has grown significantly over the last decade. One of the challenges in the translation of this burgeoning field is interpretation of the promising experimental results obtained from a variety of cell types, injury models and techniques. Although these variables can become barriers to a collective understanding and to evidence-based translation, they provide crucial information that, when correctly placed, offers the opportunity for discovery. Here, we review the preclinical evidence that is currently guiding the translation of adult stem cell therapy for TBI.

Physical activity and survival after a first myocardial infarction: The Corpus Christi Heart Project

Previous studies have demonstrated that habitual physical activity is associated with a reduced risk of incident coronary heart disease (CHD). However, the role of physical activity in lowering the risk of all-cause mortality, CHD mortality, reinfarction, or receipt of a revascularization procedure after a first myocardial infarction (MI) remains unresolved, particularly in minority populations. To investigate the associations between physical activity and risk of all-cause mortality, CHD mortality, reinfarction, and receipt of a revascularization procedure, this study was conducted among Mexican-American and non-Hispanic white women and men who survived a first MI. The Corpus Christi Heart Project, a population-based cardiovascular surveillance study, provide data which included vital status, survival time, medical history, CHD risk factor information, including level of physical activity among Mexican-American and non-Hispanic white adults who had experienced a first MI between May, 1988 and April, 1990. MI patients were interviewed at baseline and annually thereafter until their death or through May, 1995. A categorical variable was created to reflect change in level of physical activity following the first MI; categories included (1) sedentary with no change, (2) decreased activity, (3) increased activity, and (4) moderate activity with no change (the referent group). Proportional hazards regression analyses were used to assess the relationship of level of physical activity and risk of death, reinfarction, or receipt of a revascularization procedure adjusting for age, sex, ethnicity, severity of MI, and CHD risk factor status. Over a 7-year follow-up period, the relative risk (95% confidence intervals) of all-cause mortality was 4.67 (2.27, 9.60) for the sedentary-no change group, 2.33 (0.96, 5.67) for the decreased activity group, and 0.52 (0.11, 2.41) for the increased activity group. The relative risk of CHD mortality was 6.92 (2.05, 23.34) for the sedentary-no change group, 2.40 (0.55, 10.51) for the decreased activity group, and 1.58 (0.26, 9.65) for the increased activity group. The relative risk for reinfarction was 2.50 (1.52, 4.10) for the sedentary-no change group, 2.26 (1.24, 4.12) for the decreased activity group, and 0.52 (0.21, 1.32) for the increased activity group. Finally, the relative risk for receipt of a revascularization procedure was 0.65 (0.39, 1.07) for the sedentary-no change group, 0.45 (0.22, 0.92) for the decreased activity group, and 1.01 (0.51, 2.02) for the increased activity group. No interactions were observed for ethnicity or severity of first MI. These results are consistent with the hypothesis that moderate physical activity is independently associated with a lower risk of all-cause mortality, CHD mortality, and reinfarction, but not revascularization, among Mexican-American and non-Hispanic white, female and male, first MI patients. These results also support the current recommendation that physical activity plays an important role in the secondary prevention of CHD. ^

Thrombolytic therapy for acute myocardial infarction: A cohort study to evaluate a selective approach to invasive diagnosis and therapy. The Corpus Christi Heart Project

Coronary perfusion with thrombolytic therapy and selective reperfusion by percutaneous transluminal coronary angioplasty (PTCA) were examined in the Corpus Christi Heart Project, a population-based surveillance program for hospitalized acute myocardial infarction (MI) patients in a biethnic community of Mexican-Americans (MAs) and non-Hispanic whites (NHWs). Results were based on 250 (12.4%) patients who received thromobolytic therapy in a cohort of 2011 acute MI cases. Out of these 107 (42.8%) underwent PTCA with a mean follow-up of 25 months. There were 186 (74.4%) men and 64 (25.6%) women; 148 (59.2%) were NHWs, 86 (34.4%) were MAs. Thrombolysis and PTCA were performed less frequently in women than in men, and less frequently in MAs than in NHWs.^ According to the coronary reperfusion interventions used, patients were divided in two groups, those that received no-PTCA (57.2%) and the other that underwent PTCA (42.8%) after thrombolysis. The case-fatality rate was higher in no-PTCA patients than in the PTCA (7.7% versus 5.6%), as was mortality at one year (16.2% versus 10.5%). Reperfusion was successful in 48.0% in the entire cohort and (51.4% versus 45.6%) in the PTCA and no-PTCA groups. Mortality in the successful reperfusion patients was 5.0% compared to 22.3% in the unsuccessful reperfusion group (p = 0.00016, 95% CI: 1.98-11.6).^ Cardiac catheterization was performed in 86.4% thrombolytic patients. Severe stenosis ($>$75%) obstruction was present most commonly in the left descending artery (52.8%) and in the right coronary artery (52.8%). The occurrence of adverse in-hospital clinical events was higher in the no-PTCA as compared to the PTCA and catheterized patients with the exception of reperfusion arrythmias (p = 0.140; Fisher's exact test p = 0.129).^ Cox regression analysis was used to study the relationship between selected variables and mortality. Apart from successful reperfusion, age group (p = 0.028, 95% CI: 2.1-12.42), site of acute MI index (p = 0.050) and ejection-fraction (p = 0.052) were predictors of long-term survival. The ejection-fraction in the PTCA group was higher than (median 78% versus 53%) in the no-PTCA group. Assessed by logistic regression analysis history of high cholesterol ($>$200mg/dl) and diabetes mellites did have significant prognostic value (p = 0.0233; p = 0.0318) in long-term survival irrespective of treatment status.^ In conclusion, the results of this study support the idea that the use of PTCA as a selective intervention following thrombolysis improves survival of patients with acute MI. The use of PTCA in this setting appears to be safe. However, we can not exclude the possibility that some of these results may have occurred due to the exclusion from PTCA of high risk patients (selection bias). ^