Garry Chernenko

Efficacy of Rehabilitative Experience Declines with Time after Focal Ischemic Brain Injury

To maximize the effectiveness of rehabilitative therapies after stroke, it is critical to determine when the brain is most responsive (i.e., plastic) to sensorimotor experience after injury and to focus such efforts within this period. Here, we compared the efficacy of 5 weeks of enriched rehabilitation (ER) initiated at 5 d (ER5), ER14, or ER30 after focal ischemia, as judged by functional outcome and neuromorphological change. ER5 provided marked improvement in skilled forelimb reaching ability and ladder-rung- and narrow-beam-walking tasks and attenuated the stroke-induced reliance on the unaffected forepaw for postural support. ER14 provided improvement to a somewhat lesser extent, whereas recovery was diminished after ER30 such that motor function did not differ from ischemic animals exposed to social housing. To examine potential neural substrates of the improved function, we examined dendritic morphology in the undamaged motor cortex because our previous work (Biernaskie and Corbett, 2001) suggested that recovery was associated with enhanced dendritic growth in this region. ER5 increased the number of branches and complexity of layer V neurons compared with both social housing and control animals. Dendritic arbor after ER14 (although increased) and ER30 did not differ from those exposed to social housing. These data suggest that the poststroke brain displays heightened sensitivity to rehabilitative experience early after the stroke but declines with time. These findings have important implications for rehabilitation of stroke patients, many of whom experience considerable delays before therapy is initiated.

Human BAG-1/RAP46 protein is generated as four isoforms by alternative translation initiation and overexpressed in cancer cells

Previously, a Bcl-2-interacting protein, BAG-1, was cloned from mouse cells and was shown to interact with several other proteins and to be important for inhibition of apoptosis. Human BAG-1 (hBAG-1) cDNA, recently isolated by us and two other groups, has been shown to be identical to a hormone receptor-binding protein, RAP46. However, different molecular masses of hBAG-1 protein products were noted by these three groups. Here we demonstrated that hBAG-1 protein was expressed as four isoforms, designated p50, p46, p33 and p29, with apparent molecular masses of 50 kDa, 46 kDa, 33 kDa and 29 kDa, respectively. Deletion, site-directed mutagenesis and in vitro transcription/translation analysis showed that the four protein products of hBAG-1 were expressed by alternative initiation from four different start codons through a leaky scanning mechanism. Furthermore, we demonstrated that the distinct forms of hBAG-1 have different subcellular localizations, suggesting that they may have distinct functions in the cells. Characterization of hBAG-1 RNA and protein also showed that hBAG-1 was overexpressed in human cervical, breast and lung cancer cell lines. Taken together, these data clarify the conflicting observations reported in the literature and suggest that hBAG-1 is expressed as four forms of protein products, which may play a differential role in apoptosis and oncogenesis of human cells.

A critical threshold of rehabilitation involving brain-derived neurotrophic factor is required for poststroke recovery.

Background. Enriched rehabilitation (ER; environmental enrichment plus skilled reaching) improves recovery after middle cerebral artery occlusion (MCAo) in rats. Fundamental issues such as whether ER is effective in other models, optimal rehabilitation intensity, and underlying recovery mechanisms have not been fully assessed. Objective. The authors tested whether the efficacy of ER varies with ischemia model and assessed the importance of rehabilitation intensity and brain-derived neurotrophic factor (BDNF) in recovery. Methods. Rats in experiment 1 received 8 weeks of ER or remained in standard housing. Functional outcome was assessed with the staircase and cylinder tasks. Surprisingly, ER provided no functional benefit in any model. In this experiment, ER was delivered during the light phase, whereas other studies delivered ER in the dark phase of the light cycle. It was hypothesized that in the light, rats engaged in less rehabilitation or alternatively that BDNF was lower. Experiment 2 tested these hypotheses. Following MCAo, rats received ER in either the light or dark phase of the light cycle. Functional outcome was assessed and BDNF levels were measured in the motor cortex and hippocampus. Results. Recovery was accompanied by increased BDNF. This occurred only in rats that received ER in the dark and these animals reached more than those in the light condition. Conclusions. Data suggest that there is a critical threshold of rehabilitation, below which recovery will not occur, and that BDNF mediates functional recovery. The use of intensive rehabilitation therapies for stroke patients is strongly supported.

Expression of BAG-1 in Invasive Breast Carcinomas

PURPOSE The purpose of this study was to retrospectively evaluate the expression of BAG-1 in invasive breast carcinomas. The intensity and subcellular distribution of BAG-1 expression was correlated with conventional prognostic factors and with disease-free and overall survival. PATIENTS AND METHODS One hundred forty patients diagnosed with invasive breast cancer in St. Johns, Newfoundland, between 1986 and 1996 were included in the study. The median follow-up of the study was 8 years. Expression of BAG-1 was determined by immunohistochemical staining of paraffin-embedded breast tumor tissues. RESULTS Of the 140 breast carcinomas examined, 77.1% were positive for BAG-1 expression. Except for differentiation, no correlation was observed between BAG-1 expression and conventional prognostic factors such as age, histology, stage, and estrogen and progesterone receptor status. In multivariate analysis, BAG-1 expression was significantly associated with shorter disease-free (P =.0052) and overall survival (P =.0033). Patients whose tumors expressed nuclear BAG-1 tended to have a shorter disease-free (63 v 84 months; P = 0.4493) and overall (69 v 99 months, P =.1009) survival. CONCLUSION BAG-1 is overexpressed in the majority of invasive breast carcinomas. Although BAG-1 did not correlate with conventional prognostic factors, its overexpression, especially the nuclear expression, may be associated with a shorter disease-free and overall survival. Our preliminary data strongly indicate that further investigation is warranted to define the role of BAG-1 as an independent prognostic factor in patients with newly diagnosed breast cancer.

Prognostic significance of BAG-1 expression in nonsmall cell lung cancer.

The purpose of this study was to evaluate the expression of BAG‐1 in a cohort of patients with nonsmall cell lung cancer (NSCLC). The intensity and subcellular distribution of BAG‐1 expression were correlated with overall survival. Tumor samples were collected from 85 patients diagnosed with NSCLC between 1993–1995 in St. Johns, Newfoundland. Expression of BAG‐1 was determined by immunohistochemistry using polyclonal anti‐BAG‐1 antibody. There was significant variation in the immunohistochemical staining patterns of BAG‐1, including nonstaining and staining of either the cytoplasm, nucleus or both. Univariate Cox regression analysis showed that those patients whose tumor overexpressed BAG‐1 had a significant reduction in the risk of death (hazard ratio = 0.53, p = 0.03). The survival advantage of patients with BAG‐1 overexpression tumor was also demonstrated by Kaplan‐Meier analysis and log‐rank tests (median survival 30.10 months versus 17.04 months, p = 0.05). In addition, multivariate Cox regression analysis showed that patients whose tumor exhibited intense cytoplasmic staining had a further reduction of the risk of death (hazard ratio = 0.42, p = 0.03) and this effect was independent of age, stage and histology. All stages were included in the analysis. Our preliminary data strongly indicate that further investigation is warranted to better define the role of BAG‐1 as an independent prognostic factor in NSCLC.

Distinct BAG-1 isoforms have different anti-apoptotic functions in BAG-1-transfected C33A human cervical carcinoma cell line.

BAG-1 protein can be expressed as four isoforms of 50, 46, 33 and 29 kDa with different subcellular localizations, which may have different functions in anti-apoptosis, but the exact mechanism remains unclear. We constructed BAG-1 full length and deletion mutated plasmids in a pCR3.1 vector and established stable transfections of BAG-1 isoforms in low BAG-1 expressing C33A cells. Treatment of the transfected cells with cisplatin, staurosporine, paclitaxel and doxorubicine showed that BAG-1 p50, p46 and p33 isoforms enhanced the resistance to apoptosis. BAG-1 p50, p46 and p33 exhibited different degrees of apoptosis inhibition in the transfected cells and BAG-1 p46 isoform had the most pronounced effect on anti-apoptosis. BAG-1 p29 failed to protect the transfected cells from apoptosis. Resistance to apoptosis by BAG-1 isoforms was correlated with decreased caspase-3 activation. We also detected the expression of Bax, Bak, p53, Bcl-2, Bcl-XL, AIF and MRP1 by Western blots. Bcl-2 protein expression was significantly increased in p50, p46 and p33 transfected cells, while the expression of Bax, Bak, p53, Bcl-XL and MRP1 was essentially unchanged. These in vitro results suggest that distinct isoforms of BAG-1 have different anti-apoptotic functions and their functions may be correlated to increased Bcl-2 expression.

Long-term assessment of enriched housing and subventricular zone derived cell transplantation after focal ischemia in rats

The potential for using stem cells to treat stroke has garnered much interest, but stem cell therapies must be rigorously tested in animal models before transplantation studies progress to clinical trials. An enriched environment enhances transplanted subventricular zone (SVZ) cell migration and functional benefit following stroke in rats. However, the ability of SVZ cells to survive, migrate, differentiate and promote functional recovery at protracted survival times (e.g., 3 months) has not been investigated. The vasoconstrictive peptide endothelin-1 was injected adjacent to the middle cerebral artery to produce focal ischemia. Seven days later, cells derived from the SVZ of adult mice (800,000 cells/rat or vehicle injection) were transplanted into the sensory-motor cortex and striatum, and rats were then housed in enriched or standard conditions. Rats in enriched housing had access to running wheels once per week. Recovery was assessed in the forelimb-use asymmetry task (cylinder) at 1, 2, or 3 months after transplantation immediately prior to euthanasia. Transplanted cell survival and migration were quantified using stereology. Cell phenotype was determined with immunohistochemistry and confocal microscopy. Enriched housing did not enhance survival or migration of transplanted SVZ cells at protracted survival times, and the majority (~99%) of cells died within 2 months of transplantation. Cell survival was significantly, and negatively, correlated with microglial activation. Many surviving cells expressed an astrocytic phenotype. Functional recovery was not improved at any time. Therapies involving transplantation of SVZ cells following stroke must be further optimized in order to enhance long-term cell survival and thereby maximize functional benefit.

BAG-1 Expression Correlates with Bcl-2, p53, Differentiation, Estrogen and Progesterone Receptors in Invasive Breast Carcinoma

BAG-1, a recently identified anti-apoptotic protein, is overexpressed in the majority of invasive breast carcinomas. Overexpression of BAG-1 is important for both multi-step oncogenesis and resistance of cancer cells to apoptosis induced by DNA-damaging alkylating agents. BAG-1 protein species are localized differentially; nuclear expression may be associated with a shorter disease-free and overall survival in early stage breast cancer, while cytoplasmic expression has been associated with longer survival in non-small cell lung cancer. Growing evidence suggests that Bcl-2 and p53 are also involved in the oncogenesis of breast cancer. Since BAG-1 interacts with Bcl-2 and is upregulated by mutant p53 in vitro, it would be interesting to determine if their expressions are correlated with each other and with other clinical prognostic factors in invasive breast cancer. To address this question we conducted a large scale retrospective study of BAG-1, Bcl-2 and p53 in 185 breast cancer patients. Our study again showed that BAG-1 is overexpressed in the majority of breast cancer patients. In addition, it demonstrated that the expression of BAG-1 correlates with that of Bcl-2, p53, differentiation, estrogen and progesterone receptors. Our clinical study supports the preclinical finding of the interaction between BAG-1 and Bcl-2, p53 and estrogen and progesterone receptors. Further experiments to explore the prognostic and therapeutic role of BAG-1 in breast cancer are warranted.

The Effects of Repeated Rehabilitation “Tune-Ups” on Functional Recovery After Focal Ischemia in Rats

Background. For most stroke survivors, rehabilitation therapy is the only treatment option available. The beneficial effects of early rehabilitation on neuroplasticity and functional recovery have been modeled in experimental stroke using a combination of enriched environment and rehabilitation. However, the impact of a secondary intervention, such as a periodic return to therapy, remains unclear. Objective. This study examines whether a return to enriched rehabilitation (ie, “tune-up”) can further promote functional recovery or produce beneficial changes in brain plasticity in the chronic phase of stroke recovery. Methods. Rats were exposed to focal ischemia (endothelin-1 applied to forelimb sensorimotor cortex and dorsolateral striatum) and allowed to recover either in standard housing or in a combination of enriched environment and rehabilitative reaching for 9 weeks. Animals were then exposed to rotating periods of standard housing (5 weeks) and intensive “tune-up” therapy consisting of various sensorimotor/cognitive activities (2 weeks). Functional recovery was assessed using the Montoya staircase, beam-traversing, and cylinder tests, and Golgi—Cox analysis was used to examine dendritic complexity in the contralesional forelimb motor cortex. Results. Although early enriched rehabilitation significantly improved sensorimotor function in both the beam and staircase tests, “tune-up” therapy had no effect on recovery. Golgi—Cox analysis revealed no effect of treatment on dendritic complexity. Conclusions. This study reaffirms the benefits of early rehabilitation for functional recovery after stroke. However, “tune-up” therapy provided no benefit in ischemic animals regardless of earlier rehabilitation experience. It is possible that alternative approaches in the chronic phase may prove more effective.

Human papillomavirus type 16–immortalized endocervical cells selected for resistance to cisplatin are malignantly transformed and have a multidrug resistance phenotype

Cis‐diamminedichloroplatinum (II) (cisplatin, CDDP) is a highly effective chemotherapeutic agent against cervical cancer, but drug resistance is a major obstacle in its clinical application. The mechanism of drug resistance in human cervical cancer is not well understood. Here, we established an in vitro endocervical, cisplatin‐resistant cell system that mimics the development of cisplatin resistance in the human cervix. Human papillomavirus (HPV) type 16–immortalized human endocervical cells (HEN‐16‐2) were treated with cisplatin, and the cisplatin‐selected cells (HEN‐16‐2/CDDP) were resistant to cisplatin, paclitaxel, actinomycin D, doxorubicin, etoposide, and 5‐fluorouracil, thus demonstrating a multidrug resistance (MDR) phenotype. Furthermore, compared with a similar passage of drug‐sensitive HEN‐16‐2 cells, HEN‐16‐2/CDDP cells exhibited the general growth characteristics of cancer cell lines: faster growth in medium containing serum and high calcium levels, higher saturation density, anchorage‐independent growth, and formation of tumors in nude mice. These results provided the first in vitro evidence that cisplatin selection can transform HPV‐immortalized endocervical cells and cause a phenotype of MDR. Int. J. Cancer 87:818–823, 2000.