Role of the cyclin dependent kinase-4 in normal and neoplastic proliferation

In the last few years, our laboratory has studied the regulatory mechanisms of proliferation and differentiation in epidermal tissues. Our results showed differences in the roles of cyclin dependent-kinases 4 and 6, and the three D-type cyclins, during normal epidermal proliferation and neoplastic development. Thus, to elucidate the role of the different cell cycle regulators, we developed transgenic mice that overexpress CDK4 (K5-CDK4), or their cognate D-type cyclins, in epithelial tissues. The most severe phenotype was observed in K5-CDK4 animals that developed dermal fibrosis, epidermal hyperplasia and hypertrophy. Forced expression of CDK4 in the epidermal basal cell layer increased the malignant conversion of skin papillomas to squamous cell carcinomas (SCC). Contrastingly, lack of CDK4 completely inhibited tumor development, suggesting that CDK4 is required in this process. Biochemical studies demonstrated that p21 Cip1 and p27Kip1 inhibitors are sequestered by CDK4 resulting in indirect activation of Cyclin E/CDK2, implicating the non-catalytic activity of CDK4 in deregulation of the cell cycle progression. ^ It has been proposed that the proliferative and oncogenic role of Myc is linked to its ability to induce the transcription of CDK4, cyclin D1, and cyclin D2 in vitro. Deregulation of Myc oncogene has been found in several human cancers. Also it has been demonstrated that CDK4 has the ability to functionally inactivate the product of the tumor suppressor gene Rb, providing a link between Myc and the CDK4/cyclin D1/pRb/p16 pathway in some malignant tumors. Here, we sought to determine the role of CDK4 as a mediator of Myc activities by developing a Myc overexpressing mouse nullizygous for CDK4. We demonstrated that lack of CDK4 results in reduced keratinocyte proliferation and epidermal thickness in K5-Myc/CDK4-null mice. In addition, complete reversion of tumor development was observed. All together, this work demonstrates that CDK4 acts as an oncogene independent of the D-type cyclin levels and it is an important mediator of the tumorigenesis induced by Myc. In addition, we showed that the sequestering activity of CDK4 is critical for the development of epidermal hyperplasia during normal proliferation, malignant progression from papillomas to squamous cell carcinomas, and tumorigenesis induced by Myc. ^

Identification and analysis of novel mitotic inhibitors of the Caenorhabditis elegans Aurora B kinase

Proper execution of mitosis requires the accurate segregation of replicated DNA into each daughter cell. The highly conserved mitotic kinase AIR-2/Aurora B is a dynamic protein that interacts with subsets of cofactors and substrates to coordinate chromosome segregation and cytokinesis in Caenorhabdiris elegans. To identify components of the AIR-2 regulatory pathway, a genome-wide RNAi-based screen for suppressors of air-2 temperature-sensitive mutant lethality was conducted. Here, I present evidence that two classes of suppressors identified in this screen are bona fide regulators of the AIR-2 kinase. The strongest suppressor cdc-48.3, encodes an Afg2/Spaf-related Cdc48-like AAA+ ATPase that regulates AIR-2 kinase activity and stability during C. elegans embryogenesis. Loss of CDC-48.3 suppresses the lethality of air-2 mutant embryos, marked by the restoration of the dynamic behavior of AIR-2 and rescue of chromosome segregation and cytokinesis defects. Loss of CDC-48.3 leads to mitotic delays and abnormal accumulation of AIR-2 during late telophase/mitotic exit. In addition, AIR-2 kinase activity is significantly upregulated from metaphase through mitotic exit in CDC-48.3 depleted embryos. Inhibition of the AIR-2 kinase is dependent on (1) a direct physical interaction between CDC-48.3 and AIR-2, and (2) CDC-48.3 ATPase activity. Importantly, the increase in AIR-2 kinase activity does not correlate with the stabilization of AIR-2 in late mitosis. Hence, CDC-48.3 is a bi-functional inhibitor of AIR-2 that is likely to act via distinct mechanisms. The second class of suppressors consists of psy-2/smk-1 and pph-4.1, which encode two components of the conserved PP4 phosphatase complex that is essential for spindle assembly, chromosome segregation, and overall mitotic progression. AIR-2 and its substrates are likely to be targets of this complex since mitotic AIR-2 kinase activity is significantly increased during mitosis when either PSY-2/SMK-1 or PPH-4.l is depleted. Altogether, this study demonstrates that during the C. elegans embryonic cell cycle, regulators including the CDC-48.3 ATPase and PP4 phosphatase complex interact with and control the kinase activity, targeting behavior and protein stability of the Aurora B kinase to ensure accurate and timely progression of mitosis. ^

Identification of essential apoptotic genes in Drosophila

Apoptosis is a normal physiological cell suicide process which is essential for tissue homeostasis and normal development of metazoans. Misregulation of apoptosis is associated with many developmental defects and human diseases. The genes involved in the regulation and execution of apoptosis are highly conserved in humans and flies. Caspases are the executioners of cell suicide. Because of the unavailability of specific fly mutants, the developmental function of many caspase genes and genetic relationship between caspases and apoptotic components were undefined in Drosophila. We isolated several mutant alleles of the initiator caspase gene dronc, the effector casase drICE, and the Mediator component Cyclin C from the GMR-hid eyFLP/FRT screens which is designed to isolate mutants of recessive cell death genes in Drosophila melanogaster. Characterization of these mutants defined that they are essential for developmental cell death in Drosophila. dronc is required for most, but not all, cell death in Drosophila. drICE is required for apoptosis in many cells and it shares redundancy with another effector caspase gene, dcp-1, in a subset of cells in Drosophila. The genetic relationship between caspases and other apoptotic components was established through mutant analysis. We found that the pro-apoptotic protein Hid induces transcription of the initiator caspase gene dronc and the GMR-induced dronc transcripts are dependent on activated effector casapses, revealing a novel regulatory mechanism to promote caspase activity in Drosophila. Cyclin C and its kinase partner Cdk8 are required for prompt transcriptional induction of dronc in cell killing contexts. In short, we define the essential pro-apoptoic function of dronc, drICE, and Cyclin C in Drosophila and reveal a novel mechanism for regulation of dronc transcription. In the long run, these studies will help us decipher the complicated regulatory mechanism of cell death in humans. ^

A costing exercise of provision of prevention of HIV transmission from mother to child services in Vietnam

Objectives. This dissertation focuses on estimating the cost of providing a minimum package of prevention of mother-to-child HIV transmission (PMTCT) in Vietnam from a societal perspective and discussing the issues of scaling-up the minimum package nationwide. ^ Methods. Through collection of cost-related data of PMTCT services at 22 PMTCT sites in 5 provinces (Hanoi, Quang Ninh, Thai Nguyen, Hochiminh City, and An Giang) in Vietnam, the research investigates the item cost of each service in minimum PMTCT packages and the actual cost per PMTCT site at different organizational levels including central, provincial, and district. Next, the actual cost per site at each organizational level is standardized by adjusting for HIV prevalence rate to arrive at standardized costs per site. This study then uses the standardized costs per site to project, by different scenarios, the total cost to scale-up the PMTCT program in Vietnam. ^ Results. The cost for HIV tests, infant formula, and salary of health workers are consistently found to be the biggest expenditures in the PMTCT minimum package program across all organizational levels. Annual cost for drugs for prophylaxis treatment, operating and capital, and training costs are not substantial (less than 5% of total costs at all levels). The actual annual estimated cost for a PMTCT site at the central level is nearly VND 1.9 billion or US$ 107,650 (exchange rate US$ 1 = VND 17,500) while the annual cost for a provincial site is VND 375 million or US$ 21,400. The annual cost for a district site is VND 139 million (∼US$ 8,000). ^ The estimated total annual cost to roll out the PMTCT minimum package to the 5 studied provinces is approximately US$ 1.1 million. If the PMTCT program is to be scaled-up to 14 provinces until 2008 and up to 40 provinces through the end of 2010 as planned by the Ministry of Health, it would cost the health system an approximate annual amount of US$ 2.1 million and US$ 5.04 million, respectively. The annual cost for scaling-up the PMTCT minimum package nationwide is around US$ 7.6 million. Meanwhile, the total annual cost to implement PMTCT minimum packages to achieve PMTCT national targets in 2010 (providing counseling service to 90% of all pregnant women; 60% of them will receive HIV tests and 100% of HIV (+) mother and their newborn will receive prophylaxis treatment) would be US$ 6.1 million. ^ Recommendations. This study recommends: (1) the Ministry of Health of Vietnam should adjust its short-term national targets to a more feasible and achievable level given the current level of available resources; (2) a detailed budget for scaling-up the PMTCT program should be developed together with the national PMTCT action plan; (3) the PMTCT scaling-up plan developed by the Ministry of Health should focus on coverage of high prevalence population and quality of services provided rather than number of physical provinces reached; (4) exclusive breastfeeding strategy should be promoted as part of the PMTCT program; and (5) for a smooth and effective rolling out of PMTCT services nationwide, development of a national training plan and execution of this plan must precede any other initiations of the PMTCT scaling-up plan. ^

An intervention mapping approach: Identification of human trafficking victims by health professionals

Background. Today modern day slavery is known as human trafficking and is a growing pandemic that is a grave human rights violation. Estimates suggest that 12.3 million people are working under conditions of force, fraud or coercion. Working toward eradication is a worthy effort; it would free millions of humans from slavery, mostly women and children, as well as uphold basic human rights. One tactic to eradicating human trafficking is to increase identification of victims among those likely to encounter victims of human trafficking.^ Purpose. This study aims to develop an intervention that improves certain stakeholders' ability, in the health clinic setting, to appropriately identify and report victims of human trafficking to the National Human Trafficking Resource Center.^ Methods. The Intervention Mapping (IM) process was used by program planners to develop an intervention for health professionals. This methodology is a six step process that guides program planners to develop an intervention. Each step builds on the others through the execution of a needs assessment, and the development of matrices based on performance objectives and determinants of the targeted health behavior. The end product results in an ecological, theoretical, and evidence based intervention.^ Discussion. The IM process served as a useful protocol for program planners to take an ecological approach as well as incorporate theory and evidence into the intervention. Consultation with key informants, the planning group, adopters, implementers, and individuals responsible for institutionalization also contributed to the practicality and feasibility of the intervention. Program planners believe that this intervention fully meets recommendations set forth in the literature.^ Conclusions. The intervention mapping methodology enabled program planners to develop an intervention that is appropriate and acceptable to the implementer and the recipients.^

Molecular mechanisms involved in the regulation of cell polarity and genome stability by the p21 -activated kinase, Shk1, in the fission yeast, Schizosaccharomyces pombe

The essential p21-activated kinase (PAK), Shk1, is a critical component of a Ras/Cdc42/PAK complex required for cell viability, normal cell polarity, proper regulation of cytoskeletal dynamics, and sexual differentiation in the fission yeast, Schizosaccharomyces pombe. While cellular functions of PAKs have been described in eukaryotes from yeasts to mammals, the molecular mechanisms of PAK regulation and function are poorly understood. This study has characterized a novel Shk1 inhibitor, Skb15, and, in addition, identified the cell polarity regulator, Tea1, as a potential biological substrate of Shk1 in S. pombe. Skb15 is a highly conserved WD repeat protein that was discovered from a two-hybrid screen for proteins that interact with the catalytic domain of Shk1. Molecular data indicate that Skb15 negatively regulates Shk1 kinase activity in S. pombe cells. A null mutation in the skb15 gene is lethal and results in deregulation of actin polymerization and localization, microtubule biogenesis, and the cytokinetic machinery, as well as a substantial uncoupling of these processes from the cell cycle. Loss of Skb15 function is suppressed by partial loss of Shk1, demonstrating that negative regulation of Shk1 by Skb15 is required for proper execution of cytoskeletal remodeling and cytokinetic functions. A mouse homolog of Skb15 can substitute for its counterpart in fission yeast, demonstrating that Skb15 protein function has been substantially conserved through evolution. ^ Our laboratory has recently demonstrated that Shk1, in addition to regulating actin cytoskeletal organization, is required for proper regulation of microtubule dynamics in S. pombe cells. The Shk1 protein localizes to interphase and mitotic microtubules, the septum-forming region, and cell ends. This pattern of localization overlaps with that of the cell polarity regulator, Tea1, in S. pombe cells. The tea1 gene was identified by Paul Nurse's laboratory from a screen for genes involved in the control of cell morphogenesis in S. pombe. In contrast to wild type S. pombe cells, which are rod shaped, tea1 null cells are often bent and/or branched in shape. The Tea1 protein localizes to the cell ends, like Shk1, and the growing tips of interphase microtubules. Thus, experiments were performed to investigate whether Tea1 interacts with Shk1. The tea1 null mutation strongly suppresses the loss of function of Skb15, an essential inhibitor of Shk1 function. All defects associated with the skb15 mutation, including defects in F-actin organization, septation, spindle elongation, and chromosome segregation, are suppressed by tea1Δ, suggesting that Tea1 may function in these diverse processes. Consistent with a role for Tea1 in cytokinesis, tea1Δ cells have a modest cell separation defect that is greatly exacerbated by a shk1 mutation and, like Shk1, Tea1 localizes to the septation site. Molecular analyses showed that Tea1 phosphorylation is significantly dependent on Shk1 function in vivo and that bacterially expressed Tea1 protein is directly phosphorylated by recombinant Shk1 kinase in vitro. Taken together, these results identify Tea1 as a potential biological substrate of Shk1 in S. pombe. ^ In summary, this study provides new insights into a conserved regulatory mechanism for PAKs, and also begins to uncover the molecular mechanisms by which the Ras/Cdc42/PAK complex regulates the microtubule and actin cytoskeletons and cell growth polarization in fission yeast. ^