Role of stress myocardial perfusion scan in pre-operative evaluation of cancer patients undergoing non-cardiac surgery

Background. Cardiac risk assessment in cancer patients has not extensively been studied. We evaluated the role of stress myocardial perfusion imaging (MPI) in predicting cardiovascular outcomes in cancer patients undergoing non-cardiac surgery. ^ Methods. A retrospective chart review was performed on 507 patients who had a MPI from 01/2002 - 03/2003 and underwent non-cardiac surgery. Median follow-up duration was 1.5 years. Cox proportional hazard model was used to determine the time-to-first event. End points included total cardiac events (cardiac death, myocardial infarction (MI) and coronary revascularization), cardiac death, and all cause mortality. ^ Results. Of all 507 MPI studies 146 (29%) were abnormal. There were significant differences in risk factors between normal and abnormal MPI groups. Mean age was 66±11 years, with 60% males and a median follow-up duration of 1.8 years (25th quartile=0.8 years, 75th quartile=2.2 years). The majority of patients had an adenosine stress study (53%), with fewer exercise (28%) and dobutamine stress (16%) studies. In the total group there were 39 total cardiac events, 31 cardiac deaths, and 223 all cause mortality events during the study. Univariate predictors of total cardiac events included CAD (p=0.005), previous MI (p=0.005), use of beta blockers (p=0.002), and not receiving chemotherapy (p=0.012). Similarly, the univariate predictors of cardiac death included previous MI (p=0.019) and use of beta blockers (p=0.003). In the multivariate model for total cardiac events, age at surgery (HR 1.04, p=0.030), use of beta blockers (HR 2.46; p=0.011), dobutamine MPI (HR 3.08; p=0.018) and low EF (HR 0.97; p=0.02) were significant predictors of worse outcomes. In the multivariate model for predictors of cardiac death, beta blocker use (HR=2.74; p=0.017) and low EF (HR=0.95; p<0.003) were predictors of cardiac death. The only univariate MPI predictor of total cardiac events was scar severity (p=0.005). While MPI predictors of cardiac death were scar severity (p= 0.001) and ischemia severity (p=0.02). ^ Conclusions. Stress MPI is a useful tool in predicting long term outcomes in cancer patients undergoing surgery. Ejection fraction and severity of myocardial scar are important factors determining long term outcomes in this group.^

A Pre-Clinical Assessment of Minocycline for Treatment of Chronic Neuropathic Pain after Spinal Cord Injury

Patients living with a spinal cord injury (SCI) often develop chronic neuropathic pain (CNP). Unfortunately, the clinically approved, current standard of treatment, gabapentin, only provides temporary pain relief. This treatment can cause numerous adverse side effects that negatively affect the daily lives of SCI patients. There is a great need for alternative, effective treatments for SCI-dependent CNP. Minocycline, an FDA-approved antibiotic, has been widely prescribed for the treatment of acne for several decades. However, recent studies demonstrate that minocycline has neuroprotective properties in several pre-clinical rodent models of CNS trauma and disease. Pre-clinical studies also show that short-term minocycline treatment can prevent the onset of CNP when delivered during the acute stage of SCI and can also transiently attenuate established CNP when delivered briefly during the chronic stage of SCI. However, the potential to abolish or attenuate CNP via long-term administration of minocycline after SCI is unknown. The purpose of this study was to investigate the potential efficacy and safety of long-term administration of minocycline to abolish or attenuate CNP following SCI. A severe spinal contusion injury was administered on adult, male, Sprague-Dawley rats. At day 29 post-injury, I initiated a three-week treatment regimen of daily administration with minocycline (50 mg/kg), gabapentin (50 mg/kg) or saline. The minocycline treatment group demonstrated a significant reduction in below-level mechanical allodynia and above- level hyperalgesia while on their treatment regimen. After a ten-day washout period of minocycline, the animals continued to demonstrate a significant reduction in below-level mechanical allodynia and above-level hyperalgesia. However, minocycline-treated animals exhibited abnormal weight gain and hepatotoxicity compared to gapabentin-treated or vehicle-treated subjects.The results support previous findings that minocycline can attenuate CNP after SCI and suggested that minocycline can also attenuate CNP via long-term delivery of minocycline after SCI (36). The data also suggested that minocycline had a lasting effect at reducing pain symptoms. However, the adverse side effects of long-term use of minocycline should not be ignored in the rodent model. Gabapentin treatment caused a significant decrease in below-level mechanical allodynia and below-level hyperalgesia during the treatment regimen. Because gabapentin treatment has an analgesic effect at the concentration I administered, the results were expected. However, I also found that gabapentin-treated animals demonstrated a sustained reduction in pain ten days after treatment withdrawal. This result was unexpected because gabapentin has a short half-life of 1.7 hours in rodents and previous studies have demonstrated that pre-drug pain levels return shortly after withdrawal of treatment. Additionally, the gabapentin-treated animals demonstrated a significant and sustained increase in rearing events compared with all other treatment groups which suggested that gabapentin treatment was not only capable of reducing pain long-term but may also significantly improve trunk stability or improve motor function recovery.

The role of post-translation regulation of Twist expression in the tumor progression of squamous cell carcinoma of head and neck

Squamous cell carcinoma of head and neck (SCCHN) is the tenth most common cancer in the world. Unfortunately, the survival of patients with SCCHN has not improved in the last 40 years. Therefore new targets for therapy are needed, and to this end we are studying signaling pathways activated by IL-6 which we have found stimulates cell migration and soft agar growth in SCCHN. Our data show that IL-6 increases TWIST expression in a transcription-independent mechanism in many SCCHN cell lines. Further investigation reveals TWIST can be phosphorylated upon IL-6 treatment. By computation prediction (http://scansite.mit.edu/motifscan_seq.phtml ), we found that TWIST has a putative phosphorylation site for casein kinase 2 (CK2) suggesting that this kinase could serve as a link between IL-6 stimulation and Twist stability. To test this hypothesis, we used a CK2 inhibitor and shRNA to CK2 and found that these interventions inhibited IL-6 stimulation of TWIST stability. In addition, mutation of the putative CK2 phosphorylation site (S18/S20A) in TWIST decreased the amount of phospho-ATP incorporated by TWIST in an in vitro kinase assay, and altered TWIST stability. In Boyd chamber migration assay and wound-healing assay, the CK2 inhibitor, DMAT, was found to decrease the motility of IL-6 stimulated SCCHN cells and over expression of either a wild-type or the hyperphosphorylated mimicking mutant S18/20D –Twist rather than the hypo-phosphorylated mimicking mutant S18/20A-Twist can promote SCCHN cell motility.To our knowledge, this is the first report to identify the importance of IL-6 stimulated CK2 phosphorylation of TWIST in SCCHN. As CK2 inhibitors are currently under phase I clinical trials, our findings indicate that CK2 may be a viable therapeutic target in SCCHN. Therefore, further pre-clinical studies of this inhibitor are underway.

Tumor immunization strategies for enhancing the graft versus tumor activity of allogeneic bone marrow transplantation

Allogeneic bone marrow transplantation (BMT) is known to induce a beneficial anti-tumor immune response called graft-versus-tumor (GVT) activity. However, GVT activity is closely associated with graft-versus-host disease (GVHD), a potentially fatal immune response against antigens on normal recipient tissues. The T-cell populations mediating these two processes are often overlapping, but studies have shown that some donor T-cells can be tumor-specific. Therefore, the goal of this study was to develop strategies for preferentially activating donor T-cells capable of mediating GVT activity but not GVHD. The three hypotheses tested were: (1) Pre-transplant immunization of BMT donors with a recipient-derived tumor cell vaccine will induce a relative increase in GVT activity as compared to GVHD. (2) Post-transplant tumor immunization of BMT recipients will enhance GVT activity without exacerbating GVHD. (3) Pre-transplant immunization of BMT donors against a tumor-specific antigen will enhance GVT activity without exacerbating GVHD. ^ To test the first two hypotheses, C3H.SW mice (MHC-matched donors) were immunized with a C57BL/6 (recipient)-derived tumor cell vaccine (leukemia or fibrosarcoma) prior to BMT, or recipients were immunized starting one month after BMT. Both donor and recipient immunization led to a significant increase in GVT activity (enhanced recipient survival and decreased tumor growth). However, donor immunization also increased fatal GVHD, which was at least partially due to activation of alloreactive T-cells recognizing the immunodominant minor histocompatibility antigen B6dom1. GVT immunity following recipient immunization was not associated with an exacerbation of GVHD or a response to B6dom1. ^ To test the third hypothesis, influenza nucleoprotein (NP) was used as a model tumor antigen. C3H.SW donors were immunized against NP prior to BMT, which led to a significant increase in GVT activity. Although recipients were not completely protected against growth of antigen loss variant tumors, there was no increase in GVHD. ^ In conclusion, (1) immunization of allogeneic BMT donors with a recipient-derived tumor cell vaccine substantially increases GVT activity but also exacerbates GVHD, (2) post-transplant tumor immunization of allogeneic BMT recipients significantly increases GVT activity and survival without exacerbating GVHD, and (3) immunization of allogeneic BMT donors against a tumor-specific antigen significantly enhances GVT activity without exacerbating GVHD. ^

Quality of life, symptom experience, and use of complementary and alternative medicine among women with ovarian cancer

The objectives of this dissertation were to determine the quality of life in women with ovarian cancer and the association of their physical and emotional well-being with the number of symptoms, duration of symptoms, and the scores of common symptoms of ovarian cancer; to study the prevalence of complementary and alternative medicine techniques for symptom relief and its association with the number of symptoms, age, education, insurance, comorbidity, and satisfaction with medical care they received, and their pre-diagnostic experience of symptoms.^ This study was based on a secondary data analysis of a study of early detection of ovarian cancer. A sample of 139 women with ovarian cancer was recruited and was administered a questionnaire comprised of questions on their quality of life, their symptoms and what they did about the symptoms, whether they used any complementary and alternative medicine techniques, and other medical conditions they had. Out of this sample, 53 patients underwent in-depth interviews relating to their symptoms before the diagnosis and their experiences with the health care system leading to the ovarian cancer diagnosis. ^ In article #1, ovarian cancer patients were observed to have significantly poorer quality of life on all subscales and summary scores except pain, compared to that of the general population of US women. Physical well-being scores were negatively associated with the number of symptoms before diagnosis and a significant negative association of comorbidity index was observed with physical well-being. Higher education and increase in time since diagnosis was found to have better physical scores. Emotional well-being scores showed marginally significant associations with number of symptoms and bloating. ^ In article #2, a thematic content analysis of the ovarian cancer patients’ interviews revealed that on recognition of their symptoms women first assumed their symptoms to be a normal transient occurrence due to a pre-existing disease condition, or due to some other disease. A series of misattributions of their symptoms on their and their doctors’ part impacted their health care seeking.In article #3, a significantly greater likelihood of CAM use with an increase in the number of symptoms was observed.^ Based on the foregoing results, it is important to educate women on possible signs of ovarian cancer and also to educate doctors about the results of current research regarding ovarian cancer diagnosis. This will help to avoid a delay in getting a diagnosis and improve women’s quality of life. It emphasizes the diagnosis of ovarian cancer in earlier stages by more sensitive screening techniques. This study emphasizes the importance of consideration of comorbidity in any quality of life research. Additionally, educating women in the safe use of CAM techniques carries immense significance because the efficacy and safety of many of the currently advertized CAM products has not been scientifically validated. Further research is needed to confirm the findings of this study. ^

Functional Analysis of Drosophila Integrator Complex in snRNA 3' End Processing

Uridine-rich small nuclear RNAs (U snRNAs) play essential roles in eukaryotic gene expression by facilitating the removal of introns from mRNA precursors and the processing of the replication-dependent histone pre-mRNAs. Formation of the 3’ end of these snRNAs is carried out by a poorly characterized, twelve-membered protein complex named Integrator Complex. In the effort to understand Integrator Complex function in the formation of the snRNA 3’ end, we performed a functional RNAi screen in Drosophila S2 cells to identify protein factors required for snRNA 3’ end formation. This screen was conducted by using a fluorescence-based reporter that elicits GFP expression in response to a deficiency in snRNA processing. Besides scoring the known Integrator subunits, we identified Asunder and CG4785 as additional core members of the Integrator Complex. Additionally, we also found a conserved requirement for Cyclin C and Cdk8 in both fly and human snRNA 3’ end processing. We have further demonstrated that the kinase activity of Cdk8 is critical for snRNA 3’ end processing and is likely to function independent of its well-documented function within the Mediator Cdk8 module. Taken together, this work functionally defines the Drosophila Integrator Complex and demonstrates a novel function for Cyclin C/Cdk8 in snRNA 3’ end formation. This thesis work has also characterized an important functional interaction mediated by a microdomain within Integrator subunit 12 (IntS12) and IntS1 that is required for the activity of the Integrator Complex in processing the snRNA 3’ end. Through the development of a reporter-based functional RNAi-rescue assay in Drosophila S2 cells, we analyzed domains within IntS12 required for snRNA 3’ end formation. This analysis unexpectedly revealed that an N-terminal 30 amino acid region and not the highly conserved central PHD finger domain, is required for snRNA 3’ end cleavage. The IntS12 microdomain (1-45) functions autonomously, and is sufficient to interact and stabilize the putative scaffold protein IntS1. Our findings provide more details of the Integrator Complex for understanding the molecular mechanism of snRNA 3’ end processing. Moreover, these results lay the foundation for future studies of the complex through the identification of a novel functional domain within one subunit and the identification of additional subunits.

Regulations of SOX9 activity during chondrogenesis

Sox9 is a transcription factor required for chondrocyte differentiation and cartilage formation. In an effort to identify SOX9 interacting protein(s), we screened a chondrocyte cDNA library with a modified yeast two-hybrid method, Son of Sevenless (SOS) recruitment system (SRS). The catalytic subunit of cyclic AMP-dependent protein kinase A (PKA-Cα) and a new long form of c-Maf transcription factor (Lc-Maf) were found to interact specifically with SOX9. We showed here that two PKA phosphorylation consensus sites of SOX9 could be phosphorylated by PKA in vitro as well as in vivo. PKA phosphorylation of SOX9 increases its DNA binding and transcriptional activities on a Col2a1 chondrocyte-specific enhancer. Mutations of these two PKA phosphorylation sites markedly decreased the activation of SOX9 by PKA. ^ To test whether parathyroid hormone-related peptide (PTHrP) signaling results in SOX9 phosphorylation, we generated a phosphospecific antibody that specifically recognizes SOX9 that is phosphorylated at serine 181 (S 181) one of the two consensus PKA phosphorylation sites. Addition of PTHrP to COS7 cells cotransfected with SOX9 and PTH/PTHrP receptor strongly increased phosphorylation of SOX9 at S181; this phosphorylation was blocked by a PKA-specific inhibitor. In similar experiments we showed that PTHrP increased the activity of a SOX9-dependent Col2a1 enhancer. This increase in activity was abolished when a SOX9 mutant was used containing serine-to-alanine substitution in the two consensus PKA phosphorylation sites of SOX9. Using our phosphospecific SOX9 antibody we showed by immunohistochemistry of mouse embryos that Sox9 phosphorylated at S181 was localized almost exclusively in the pre-hypertrophic zone of the growth plate, an area corresponding to the major site of expression of PTH/PTHrP receptor. In contrast, no phosphorylation of Sox9 at S181 was detected in growth plates of PTH/PTHrP receptor null mutant mice. Sox9, regardless of phosphorylation state, was present in all chondrocytes of both genotypes except in hypertrophic chondrocytes. Thus, Sox9 is a target of PTHrP signaling and the PTHrP-dependent phosphorylation of SOX9 enhances its transcriptional activity. ^ In order to investigate the in vivo function of Sox9 phosphorylation by PKA, we are generating a mouse model of mutant Sox9 harboring point mutations in two PKA phosphorylation sites. Preliminary results indicated that heterozygous mice containing half amount of mutant Sox9 that can not be phosphorylated by PKA have normal skeletal phenotype and homozygous mice are being generated. ^ Lc-Maf encodes an extra ten amino acids at the carboxyl terminus of c-Maf and contains a completely different 3′ untranslated region. The interaction between SOX9 and Lc-Maf was further confirmed by co-immunoprecipitation and GST-pull down assays, which mapped the interacting domains of SOX9 to HMG DNA binding domain and that of Lc-Maf to basic leusine zipper motif. In situ hybridizations showed that RNA of Lc-Maf coexpressed with those of Sox9 and Col2a1 in areas of mesenchymal condensation during the early stages of mouse embryo development. A DNA binding site of Lc-Maf was identified at the 5′ part of a 48-bp Col2a1 enhancer element near the HMG binding site of SOX9. Lc-Maf and SOX9 synergistically activated a luciferase reporter plasmid containing a Col2al enhancer and increased the transcription of endogenous Col2a1 gene. In summary, Lc-Maf is the first identified SOX9-interating protein during chondrogenesis and may be an important activator of Col2a1 gene. ^