The p53 tumor suppressor pathway in culture and {\it in vivo\/}

p53 functions as a tumor suppressor through its ability to initiate either growth arrest or apoptosis in cells which have sustained DNA damage. p53 elicits these cellular phenotypes through its biochemical function as a transcriptional activator. By inducing the expression of a battery of target genes, p53 is able to prevent the propagation of cells with damaged DNA. However, the genes transcriptionally induced by p53 which have been identified to date do not fully explain p53 function. p53 has been demonstrated to activate genes involved in cell cycle inhibition, apoptosis and cell proliferation. The reasons for simultaneous activation of p53 targets with disparate, opposing functions are not clear, but may be due to the use of transformed cell lines in previous experiments. In the studies presented in this thesis, the pathway of p53 tumor suppression has been studied in detail in two systems chosen for their relevance to the natural cell environment. One utilizes a normal, unaltered cultured cell system; the other the whole mouse. In order to better understand the role of the known p53 targets in effecting p53 function in normal cells, early rat embryo fibroblasts were irradiated with ultraviolet light to induce DNA damage. It was discovered that p53 protein levels increased in response to irradiation. The known targets of p53, namely, $p21\sp{WAF1/CIP1},\ mdm2,\ cyclin\ G,$ and bax, were shown for the first time to have a differential temporal induction. The growth suppressor $p21\sp{WAF1/CIP1}$ was induced first, followed by cyclin G then mdm2, which is involved in proliferation through its inactivation of p53, and finally, the apoptosis promoter, bax. These findings indicated that p53 activates its target genes in a manner to allow maximum effectiveness of target function. The rat embryo fibroblasts were shown to undergo apoptosis 24 h after irradiation. Additionally, investigation of these cells for cell cycle alterations demonstrated a brief arrest in G1. In the second study, thymocytes from mice with wild type p53 were shown to undergo apoptosis and activate p53 target genes upon ionizing radiation treatment, while thymocytes from mice deficient in p53 could not. The p53 target genes mdm2 and fas were tested in vivo for their ability to mediate p53-regulated apoptosis, and were found dispensible for that cellular function. Therefore, the p53 targets identified to date do not fully explain the ability of p53 to function as a tumor suppressor. Potentially, functional redundancy between the known targets would account for the data seen in these experiments. Additionally, identification of additional target genes should add further understanding of the p53 pathway of tumor suppression. ^

Role of germ line p53 mutations in aneuploidy and immortalization of dermal fibroblasts from Li-Fraumeni cancer patients

Pedigree analysis of certain families with a high incidence of tumors suggests a genetic predisposition to cancer. Li and Fraumeni described a familial cancer syndrome that is characterized by multiple primary tumors, early age of onset, and marked variation in tumor type. Williams and Strong (1) demonstrated that at least 7% of childhood soft tissue sarcoma patients had family histories that is readily explained by a highly penetrant autosomal dominant gene. To characterize the mechanism for genetic predisposition to many tumor types in these families, we have studied genetic alterations in fibroblasts, a target tissue from patients with the Li-Fraumeni Syndrome (LFS).^ We have observed spontaneous changes in initially normal dermal fibroblasts from LFS patients as they are cultured in vitro. The cells acquire an altered morphology, chromosomal anomalies, and anchorage-independent growth. This aberrant behavior of fibroblasts from LFS patients had never been observed in fibroblasts from normal donors. In addition to these phenotypic alterations, patient fibroblasts spontaneously immortalize by 50 population doublings (pd) in culture; unlike controls that remain normal and senesce by 30-35 (2). At 50 pd, immortal fibroblasts from two patients were found to be susceptible to tumorigenic transformation by an activated T24 H-ras oncogene (3). Approximately 80% of the oncogene expressing transfectants were capable of forming tumors in nude mice within 2-3 weeks. p53 has been previously associated with immortalization of cells in culture and cooperation with ras in transfection assays. Therefore, patients' fibroblast and lymphocyte derived DNA was tested for point mutations in p53. It was shown that LFS patients inherited certain point mutations in one of the two p53 alleles (4). Further studies on the above LFS immortal fibroblasts have demonstrated loss of the remaining p53 allele concomitant with escape from senescence. While the loss of the second allele correlates with immortalization it is not sufficient to transformation by an activated H-ras or N-ras oncogene. These immortal fibroblasts are resistant to tumorigenic transformation by v-abl, v-src, c-neu or v-mos oncogene; implying that additional steps are required in the tumorigenic progression of LFS patients' fibroblasts.^ References. (1) Williams et al., J. Natl. Cancer Inst. 79:1213, 1987. (2) Bischoff et al., Cancer Res. 50:7979, 1990. (3) Bischoff et al., Oncogene 6:183, 1991. (4) Malkin et al., Science 250:1233, 1990. ^

IDENTIFICATION AND CHARACTERIZATION OF DISTINCT POPULATIONS OF CLONOGENIC BONE MARROW STROMAL CELLS CAPABLE OF TRANSFERRING THE HEMATOPOIETIC MICROENVIRONMENT IN VIVO AND SUPPORTING LT-HSCs IN VITRO

Bone marrow (BM) stromal cells are ascribed two key functions, 1) stem cells for non-hematopoietic tissues (MSC) and 2) as components of the hematopoietic stem cell niche. Current approaches studying the stromal cell system in the mouse are complicated by the low yield of clonogenic progenitors (CFU-F). Given the perivascular location of MSC in BM, we developed an alternative methodology to isolate MSC from mBM. An intact ‘plug’ of bone marrow is expelled from bones and enzymatically disaggregated to yield a single cell suspension. The recovery of CFU-F (1917.95+199) reproducibly exceeds that obtained using the standard BM flushing technique (14.32+1.9) by at least 2 orders of magnitude (P<0.001; N = 8) with an accompanying 196-fold enrichment of CFU-F frequency. Purified BM stromal and vascular endothelial cell populations are readily obtained by FACS. A detailed immunophenotypic analysis of lineage depleted BM identified PDGFRαβPOS stromal cell subpopulations distinguished by their expression of CD105. Both subpopulations retained their original phenotype of CD105 expression in culture and demonstrate MSC properties of multi-lineage differentiation and the ability to transfer the hematopoietic microenvironment in vivo. To determine the capacity of either subpopulation to support long-term multi-lineage reconstituting HSCs, we fractionated BM stromal cells into either the LinNEGPDGFRαβPOSCD105POS and LINNEGPDGFRαβPOSCD105LOW/- populations and tested their capacity to support LT-HSC by co-culturing each population with either 1 or 10 HSCs for 10 days. Following the 10 day co-culture period, both populations supported transplantable HSCs from 10 cells/well co-cultures demonstrating high levels of donor repopulation with an average of 65+23.6% chimerism from CD105POS co-cultures and 49.3+19.5% chimerism from the CD105NEG co-cultures. However, we observed a significant difference when mice were transplanted with the progeny of a single co-cultured HSC. In these experiments, CD105POS co-cultures (100%) demonstrated long-term multi- lineage reconstitution, while only 4 of 8 mice (50%) from CD105NEG -single HSC co-cultures demonstrated long-term reconstitution, suggesting a more limited expansion of functional stem cells. Taken together, these results demonstrate that the PDGFRαβCD105POS stromal cell subpopulation is distinguished by a unique capacity to support the expansion of long-term reconstituting HSCs in vitro.

Understanding mental illness in Nigeria: Bringing culture and traditional medicine into mental health policy

One of the major challenges in treating mental illness in Nigeria is that the health care facilities and mental health care professionals are not enough in number or well equipped to handle the burden of mental illness. There are several barriers to treatment for individual Nigerians which include the following: such as the lack of understanding of the root causes of mental illness, lack of financial support to get mental treatment, lack of social support (family, friends, neighbors), the fear of stigmatization concerning being labeled as mentally ill or being in association with the mentally ill, and the consultation of traditional native healers who may be unknowingly prolonging illness, rather than addressing and treating them due to lack of formal education and standardization of their treatments. Another barrier is the non-health nature of the mental health services in Nigeria. Traditional healers are essentially the mental health system. The elderly, women, and children are the most vulnerable groups in times of strife and hardships. Their mental well-being must be taken into account as well as their special needs in times of personal or societal crisis. ^ Nigerian mental health policy is geared toward forming a mental health system, but in actuality only a mental illness care system is the observed result of the policy. The government of Nigeria has drafted a mental health policy, yet its actual implementation into the Nigerian health infrastructure and society waits to be materialized. The limited health legislation or policy implementations tend to favor those who have access to these urban areas and the facilities' health services. Nigerians living in rural areas are at a disadvantage; many of them may not even be aware of services available to help them understand and treat mental illness. Perhaps, government driven health interventions geared toward mental illness in rural areas would reach an underserved Nigerians and Africans in general. Issues with political instability and limited infrastructure often hinder crucial financial resources and legislation from reaching the people that are truly in need of governmental leadership in regards to mental health policy.^ Traditional healers are a severely untapped resource in the treatment of mental illness within the Nigerian population. They are abundant within Nigerian communities and are meeting a real need for the mentally ill. However, much can be done to remove the barriers that prevent the integration of traditional healers within the mental health system and improve the quality of care they administer within the population. Mental illness is almost exclusively coped with through traditional medicine practices. Mobilization and education from each strata of Nigerian society and government as well as input from the medical community can improve how traditional medicine is utilized as a treatment for clinical illness and help alleviate the heavy burden of mental illness in Nigeria. Currently, there is no existing policy making structure for a working mental health system in Nigeria, and traditional healers are not taken into account in any formulation of mental health policy. Advocacy for mental illness is severely inadequate due to fear of stigmatization, with no formally recognized national of regional mental health association.^

THE EFFECT OF RELAXIN ON BIOCHEMICAL AND MORPHOLOGICAL PARAMETERS OF RAT MYOMETRIAL CELLS IN CULTURE (CYCLIC-AMP, CELL SHAPE CHANGE)

Relaxin is able to inhibit spontaneous, oxytocin-and prostaglandin-driven uterine contractions. The intracellular mechanism of action of relaxin on uterine relaxation had previously been studied using isometrically suspended uterine strips. Since uterine strips contain stroma as well as myometrium, the changes in biochemical parameters induced by relaxin treatment may not occur in the same cell types responsible for the physical changes. In these studies, cultures of enriched populations of rat myometrial cells were used to investigate the effect of relaxin on biochemical and morphological parameters which are related to relaxation.^ Under optimal culture conditions (initial plating density 1 - 1.5 x 10('6)cells/ml, 3 ml/35 mm dish, 2 days culture), enzymatically isolated rat myometrial cells were able to respond to relaxin with cAMP elevation. Relaxin elevated cAMP levels in the presence but not the absence of 0.1 mM methylisobutylxanthine or 0.4 um forskolin in a time- and concentration-dependent manner. In contrast, isoproterenol was able to elevate cAMP levels in the presence and absence of 0.1 mM methylisobutylxanthine.^ Oxytocin treatment caused a decrease in mean cell length and area of myometrial cells in culture which could be considered analogous to contraction. Under optimal culture conditions, relaxin increased myometrial cell length and area (i.e. analogous to relaxation) of oxytocin-treated cells in a time- and concentration-dependent manner. Other relaxants such as isoproterenol and dibutyryl cAMP also increased cell length and area of oxytocin - treated myometrial cells in culture.^ Under optimal culture conditions, relaxin decreased myosin light chain kinase activity in a time-and concentration-dependent manner by increasing the K(,50) of the enzyme for calmodulin (CaM), i.e. decreasing the affinity of the enzyme for CaM. The decrease in the affinity of myosin light chain kinase for CaM may be due to the phosphorylation of the enzyme by cAMP-dependent protein kinase. Relaxin also decreased the Ca('2+)(.)CaM-independent myosin light chain kinase activity to a lesser extent than that of the Ca('2+)(.)CaM-dependent enzyme activity. This was not attributable to a decrease in the affinity of the enzyme for myosin in myometrial cells in culture, in contrast to the finding of such a change following relaxin treatment of uterine strips. Further studies are required to clarify this point.^ There was a temporal association between the effects of relaxin on elevation of cAMP levels in the presence of 0.4 uM forskolin, increase in cell length and decrease in myosin light chain kinase activity. . . . (Author's abstract exceeds stipulated maximum length. Discontinued here with permission of author.) UMI ^

Everyone is; should healthcare? Outsourcing: Issues in strategic planning, organizational culture, and organizational ethics

Expenditures for personal health services in the United States have doubled over the last decade. They continue to outpace the growth rate of the gross national product. Costs for medical care have steadily increased at an annual rate well above the rate of inflation and have gradually outstripped payers' ability to meet their premiums. This limitation of resources justifies the ongoing healthcare reform strategies to maximize utilization and minimize costs. The majority of the cost-containment effort has focused on hospitals, as they account for about 40 percent of total health expenditures. Although good patient outcomes have long been identified as healthcare's central concern, continuing cost pressures from both regulatory reforms and the restructuring of healthcare financing have recently made improving fiscal performance an essential goal for healthcare organizations. ^ The search for financial performance, quality improvement, and fiscal accountability has led to outsourcing, which is the hiring of a third party to perform a task previously and traditionally done in-house. The incomparable nature and overwhelming dissimilarities between health and other commodities raise numerous administrative, organizational, policy and ethical issues for administrators who contemplate outsourcing. This evaluation of the outsourcing phenomenon, how it has developed and is currently practiced in healthcare, will explore the reasons that healthcare organizations gravitate toward outsourcing as a strategic management tool to cut costs in an environment of continuing escalating spending. ^ This dissertation has four major findings. First, it suggests that U.S. hospitals in FY2000 spent an estimated $61 billion in outsourcing. Second, it finds that the proportion of healthcare outsourcing highly correlates with several types of hospital controlling authorities and specialties. Third, it argues that healthcare outsourcing has implications in strategic organizational issues, professionalism, and organizational ethics that warrant further public policy discussions before expanding its limited use beyond hospital “hotel functions” and back office business processes. Finally, it devises an outsourcing suitability scale that organizations can utilize to ensure the most strategic option for outsourcing and concludes with some public policy implications and recommendations for its limited use. ^

Culture and the social context of health inequalities

There has been a great deal of interest and debate recently concerning the linkages between inequality and health cross-nationally. Exposures to social and health inequalities likely vary as a consequence of different cultural contexts. It is important to guide research by a theoretical perspective that includes cultural and social contexts cross-nationally. If inequality affects health only under specific cultural conditions, this could explain why some of the literature that compares different societies finds no evidence of a relationship between inequality and health in certain countries. A theoretical framework is presented that combines sociological theory with constructs from cultural psychology in order to identify pathways that might lead from cultural dimensions to health inequalities. Three analyses are carried out. The first analysis explores whether there is a relationship between cultural dimensions at the societal level and self-rated health at the individual level. The findings suggest that different cultural norms at the societal level can produce both social and health inequalities, but the effects on health may differ depending on the socio-cultural context. The second analysis tests the hypothesis that health is affected by the density of social networks in a society, levels of societal trust, and inequality. The results suggest that commonly used measures of social cohesion and inequality may have both contextual and compositional effects on health in a large number of countries, and that societal measures of social cohesion and inequality interact with individual measures of social participation, trust, and income, moderating their effects on health. The third analysis explores whether value systems associated with vertical individualist societies may lead to health disparities because of their stigmatizing effects. I test the hypothesis that, within vertical individualist societies, subjective well-being will be affected by a social context where competition and the Protestant work ethic are valued, mediated by inequality. The hypothesis was not supported by the available cross-national data, most likely because of inadequate measures, missing data, and the small sample of vertical individualist countries. The overall findings demonstrate that cultural differences are important contextual factors that should not be overlooked when examining the causes of health inequalities. ^

Evaluation of therapist and client language in Motivational Interviewing (MI) sessions: A secondary analysis of data from the Southern Methodist Alcohol Research Trial (SMART) study

In the United States, “binge” drinking among college students is an emerging public health concern due to the significant physical and psychological effects on young adults. The focus is on identifying interventions that can help decrease high-risk drinking behavior among this group of drinkers. One such intervention is Motivational interviewing (MI), a client-centered therapy that aims at resolving client ambivalence by developing discrepancy and engaging the client in change talk. Of late, there is a growing interest in determining the active ingredients that influence the alliance between the therapist and the client. This study is a secondary analysis of the data obtained from the Southern Methodist Alcohol Research Trial (SMART) project, a dismantling trial of MI and feedback among heavy drinking college students. The present project examines the relationship between therapist and client language in MI sessions on a sample of “binge” drinking college students. Of the 126 SMART tapes, 30 tapes (‘MI with feedback’ group = 15, ‘MI only’ group = 15) were randomly selected for this study. MISC 2.1, a mutually exclusive and exhaustive coding system, was used to code the audio/videotaped MI sessions. Therapist and client language were analyzed for communication characteristics. Overall, therapists adopted a MI consistent style and clients were found to engage in change talk. Counselor acceptance, empathy, spirit, and complex reflections were all significantly related to client change talk (p-values ranged from 0.001 to 0.047). Additionally, therapist ‘advice without permission’ and MI Inconsistent therapist behaviors were strongly correlated with client sustain talk (p-values ranged from 0.006 to 0.048). Simple linear regression models showed a significant correlation between MI consistent (MICO) therapist language (independent variable) and change talk (dependent variable) and MI inconsistent (MIIN) therapist language (independent variable) and sustain talk (dependent variable). The study has several limitations such as small sample size, self-selection bias, poor inter-rater reliability for the global scales and the lack of a temporal measure of therapist and client language. Future studies might consider a larger sample size to obtain more statistical power. In addition the correlation between therapist language, client language and drinking outcome needs to be explored.^