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Biochemistry/Molecular Biology. (Abstracts-2003 Annual Meetings).

Vitamin D in The Prevention and Treatment of Prostate Cancer. Erik J. Tokar, Daniel E. Williams, and Mukta M. Webber, Departments of Zoology and Medicine, Michigan State University, East Lansing, Ml 48824-13 12

Prostate cancer, the second leading cause of death from cancer in American men, has a 20- to 30-year latency period before cancer is manifested. It may, therefore, be possible to prevent cancer development. An inverse relationship between prostate cancer mortality and UV light exposure (essential for vitamin D synthesis) has been reported. Vitamin D plays an important role in cell differentiation. We examined the ability of vitamin D3 (cholecalciferol) to inhibit or reverse cellular changes associated with carcinogenesis and cancer invasion. Nontumorigenic human prostate cell line, RWPE-1 (immortalized with HPV-18), and the malignant RWPE2-W99 cell line, derived from RWPE-1 by Ki-ras transformation, were used. The effects of cholecalciferol on both cell lines were first determined. Further, the effects on cytoskeleral proteins, matrix metalloproteinases, motility, and invasion were determined by immunostaining, SDS-PAGE zymography, and in vitro invasion assays. Preliminary results show that cholecalciferol u pregulares vitamin D and certain retinoid receptors, inhibits growth and invasion, induces differentiation, and decreases the expression of proteins associated with malignant transformation. This evidence suggests that vitamin D may be an important dietary component for cancer prevention. Its effects and mechanisms of action are being investigated.

A Human Prostate Cancer Xenograft Model in Nude Mice Mimics Disease Stages. Amanda S. Rivette, Erik J. Tokar, Daniel E. Williams, and Mukta M. Webber, Departments of Zoology and Medicine, Michigan State University, East Lansing, MI 48824-1312

Prostate cancer is the second leading cause of death from cancer in American men. In order to better understand the disease and to accelerate development of new therapies, in vivo models that reflect different disease stages are needed. A family of cell lines, that mimic multiple steps in cancer development and progression, have been developed in our laboratory. The parent, nontumorigenic, RWPE- 1 cell line was derived by immortalization with HPV- 18. The tumorigenic cell lines were derived from RWPE- 1 by transformation using different methods: RWPE2-W99 cells by Ki-ras (Carcinogenesis 1997, 1 8:1225-23 1); MNU cell lines by N-methul-N-nitrsourea (The Prostate 47[2001]:1-13), and CTPE cells by cadmium (Cancer Res. 61[2001]: 455-58). In a pilot study to develop the in vivo protocol for tumor development, RWPE2-W99 and CTPE cells were injected subcutaneously into two separate groups of immunedeficient mice and maintained for 10 weeks. Tumors and lungs were processed for histology. RWPE2-W99 cells formed small , noninvasive tumors while CTPE cells formed invasive tumors with metastasis to the lungs. Other cell lines are being tested to develop an in vivo model representing different disease stages. These cell lines provide an invaluable model system for studying tumor development and progression, and for the evaluation of agents for prevention and treatment.

Modulation of Intermediate Filament Proteins by Retinoids in Prostate Cancer. LaToyia D. Johnson, Erik J. Tokar, and Mukta M. Webber, Departments of Zoology and Medicine, Michigan State University, East Lansing, MI 48824-1312

There is an urgent need to develop strategies for prostate cancer prevention because it is the leading cancer in men in the United States. Its long latent period of 20 to 30 years provides the opportunity to prevent prostate cancer. Our specific aim is to assess the ability of retinoids, the natural and synthetic derivatives of vitamin A, to restore normal expression of the intermediate filament cytoskeletal proteins cytokeratins and vimentin. Prostatic epithelial cells, from which prostate cancer arises, express cytokeratins 8 and 18. Co-expression of vimentin and cytokeratins, not normally seen in epithelial cells, is associated with malignant transformation. Vimentin expression imparts increased motility and invasiveness to cancer cells. Extracts of untreated and retinoid-treated cells were analyzed by SDS-polyacrylamide gel electrophoresis and Western blot analysis to examine modulation of cytokeratins and vimentin. A marked decrease in vimentin expression was observed after retinoid treatment and is asso ciated with inhibition of invasion by cancer cells. These results suggest that retinoids may have important application not only in the prevention of prostate cancer but also in controlling the spread of cancer to other organs.

Identification of ACTH-Dependent, Phospho-PKA Substrates within the Nucleus. Megan Hall, Jonathan N. Winnay, and Gary Hammer, University of Michigan, Metabolism and Endocrinology; home address: 563 Mosher Jordan Hall 200 Observatory, Ann Arbor, MI 48109-2035

The POMC-derived peptide ACTH represents the primary peptide hormone responsible for steroidogenesis in the adrenal cortex. The binding of ACTH to its cognate receptor stimulates the production of cAMP and the downstream activation of cAMP-dependent protein kinase A (PKA). Analysis of the promoter regions of the genes encoding the steroid hydroxylases has led to the identification of cAMP-responsive elements, but little is known about the mechanism through which PKA mediates the transcription of these target genes. The orphan nuclear receptor steroidogenic factor-1 (SF-1) was originally identified as a key regulator of the tissue-specific expression of the steroid hydroxylases. Interestingly, SF-1 consensus binding sites map to these cAMP-responsive elements, thereby implicating SF-1 as a downstream mediator of cAMP responsiveness. In order to identify effectors downstream of PKA activation, we have utilized an antibody that is capable of recognizing serine or threonine phosphorylated PKA substrates. Experime nts utilizing this antibody have led to the identification of several ACTH-dependent, phospho-PKA substrates within the nucleus. The time-dependent increase in the phosphorylation of these proteins suggests that they may be important downstream effectors responsible for mediating the PKA-dependent activation of steroidogenic enzyme gene expression.

Generation of Human DNA Profiles by Alu-Based Multiplex PCR. David H. Kass, Eastern Michigan University, Biology Department, 316 Mark Jefferson, Ypsilanti, MI 48197; david.kass@emich.edu

Alu elements have been demonstrated to provide a unique set of DNA markers utilized in human diversity studies, paternity cases, and forensic analyses. More recent insertions of these retrotransposons into the human genome yield presence/absence variants referred to as allelic dimorphisms. Advantages of this type of marker include knowledge of an ancestral form, as well as the utilization of a simple PCR based assay. Modifications and improvements were made in the development of multiplex PCR for the analysis of several dimorphic Alu-containing loci in a single reaction. Two highly variable and reproducible multiplex reactions were developed. Concurrent use of a triplex and tetraplex reaction yielded unique DNA profiles for 46 analyzed individuals. Multiplex PCR of Alu dimorphisms provides a valuable, rapid, low-cost tool for DNA fingerprinting applications.

Substrate Specificity in the Subtilisin-like Proprotein Convertases: A Role for Hydrophobic Amino Acids Adjacent to and Within the Substrate Cleavage Recognition Sequence. Ericca Stamper, Anna Y. Fleytman, John M. Sushynski, Eric Lerche, Jeremy R. Hogg, Beth A. Ziemba, and Joseph F. Sucic, Biology Department, University of Michigan-Flint, Flint, MI 48502-1950; jsucic@umflint.edu

Subtilisin-like proprotein convertases (SPCs) are eukaryotic endoproteases. Four SPCs (futin, PACE4, PC5, and PC7) are widely expressed and play critical roles in development, homeostasis; and pathology. All substrates of the widely expressed SPCs are processed on the carboxy terminal side of the sequence N-Arginine-X-X-Arginine-C. While all substrates contain this sequence, each widely expressed SPC has unique substrate preferences and many proproteins are processed differentially by these enzymes. We observed that almost all differentially processed proproteins harbor hydrophobic residues directly adjacent to or within the cleavage sequence; in contrast, most proproteins acted upon by all widely expressed SPCs harbor hydrophilic residues in the same positions. We hypothesized that these hydrophobic residues might mediate substrate specificity in SPCs by impacting the ability of a specific SPC to recognize and bind to a specific proprotein. Site-directed mutagenesis was used to replace hydrophilic residues i n pro-von Willebrand's factor, processed by all widely expressed SPCs, with hydrophobic residues seen in proteins that are differentially processed. Results suggest that certain hydrophobic residues adjacent to and within the cleavage recognition sequences can mediate substrate specificity.

Post-Cryogenic Biochemical Comparison of Spermatozoa Membranes Isolated Blue Fox and Silver Fox. Crystal L. Comett, Robert R. Miller, Jr., Hillsdale College, Biology Department, 33 E. College, Hillsdale, MI 49242

Cryogenic protocols have been developed for the storage of silver fox (Vulpes vulpes) spermatozoa. However these same protocols and modifications of these protocols have failed to preserve spermatozoa collected from the endangered blue fox (Alopex lagopus). Differences in the cryogenic behavior of spermatozoa may be related to differences in membrane fluidity. Spermatozoa that can be cryogenically preserved may have more fluid membranes, facilitating the removal of water prior to cryogenic freezing. However, spermatozoa that cannot be cryogenically frozen may have less fluid membranes which impairs dehydration and facilitates ice-crystal damage during the cryogenic process. Since membrane fluidity is determined by the fatty acid and steroid composition of membranes, we have subjected isolated spermatozoa membranes from blue fox and silver fox to biochemical analyses. Silver fox spermatozoa membranes have significantly higher levels of docosapentaenoic acid (22:5, n-3), higher levels of membrane desmosterol, a nd higher levels of membrane cholesterol as compared to blue fox spermatozoa. Blue fox spermatozoa membranes have significantly higher levels of linoleic acid (18:3, n-3) as compared to silver fox spermatozoa.

A Novel Human Cell Model for Studies on Cancer Prevention. Antonia M. Jerkins, Erik J. Tokar, and Mukta M. Webber, Departments of Zoology and Medicine, Michigan State University, East Lansing, MI 48824-1312

Cancer development and its progression are multistep processes that occur over a period of many years. It is, therefore, important to attempt to reverse or block one or more of these steps with the goat of preventing cancer development. A unique family of cell lines that mimic many of these steps has been developed in our laboratory. A nonmalignant, epithelial, and four derived, malignant cell lines, representing multiple steps in prostate cancer development and progression, were used. Evidence suggests that vitamin D deficiency may be a risk factor for developing prostate cancer. The specific aims of this study are to examine (1) growth characteristics of the five cell lines, (2) their response to vitamin D3 (cholecalciferol) exposure, and (3) vitamin D3 effect on vimentin expression, a cytoskeletal protein associated with motility and invasion by cancer cells. A microplate, MTT assay for growth, and immunoperoxidase staining for vimentin expression were used. The nonmalignant and the least malignant cells s how slow, while the highly malignant cells show rapid growth. Response to cholecalciferol varied amongst cell lines. Cholecalciferol treatment caused a marked decrease in vimentin expression. These results indicate that vitamin D may be useful for treatment and prevention of prostate cancer.

Over-expression and Purification of the Rat Isl-1 LIM/Homeodomain Polypeptide. Jeremiah J. Frye and Richard W. Frazee, University of Michigan--Flint, Chemistry Department, Flint, MI 48502

The insulin enhancer binding protein, Isl-1, is a homeodomain (HD) containing transcription factor that regulates insulin expression in differentiated islet cells as well as important processes in the development of both islet cells and motor neurons. It possesses both a homeodomain and two LIM (lin-11, isl-1, mec-3) domains. Each LIM domain incorporates Zn via "zinc finger" like cysteinyl/hisridinyl ligation. Because of inclusion body formation during over-expression, characterization of soluble Isl-1 has been difficult. Improper folding as a result of insufficient Zn incorporation may contribute to the problem. We show that growing E. coli BL21 (DE3 )-pLysS expressing the LIM/HD polypeptide in the presence of 0.2 mM Zn[SO.sub.4] results in reasonable production of the protein in the soluble phase. Fractionation of cell extracts using a BioRex-70 resin yields a protein corresponding to the approximate molecular weight of the LIM/HD protein. It was found to elute at the same NaCl concentration, 0.45 M, as the Isl-1 homeodomain, which we have recently purified. Both the apparent molecular weight and elution profile are consistent with that expected for the LIM/HD polypeptide. As judged by SDS-PAGE and coomassie staining, the probable LIM/HD protein is greater then 80% pure. Efforts to identify and characterize the protein are ongoing.

Cysteine Residues in the Catalytic Domain of Furin are Essential for Catalytic Activity and Proper Subcellular Trafficking. Beth A. Ziemba, Kristin M. Hockman, and Joseph F. Sucic, University of Michigan-Flint, Biology Department, Flint, MI 48502-1950; jsucic@umflint.edu

Furin is a subtilisin-like endoprotease involved in processing a diverse array of proproteins within the secretory and endocytic pathways of a wide variety of organisms and mammalian cell types. Five cysteine residues within the catalytic domain are highly conserved across species (C104, 0196, C198, C226, and 0253). Molecular modeling studies have suggested four of these cysteines form two disulfide bonds necessary for furin function. We utilized site-directed mutagenesis to change each cysteine residue to a serine. Consequences of the changes were examined by coexpressing the mutant versions of furin with pro-von Willebrand's factor (pro-vWF), which is efficiently processed by wild type furin. Four mutations (0104S, C196S, C226S, and C253S) completely abolished pro-vWF processing ability, consistent with the hypothesis that critical disulfide bonds are formed between C104 and 0253 and 0196 and C226. The C198S mutation partially impaired pro-vWF processing. None of the mutant versions of furin exhibited evide nce of autocatalysis. Since autocatalytic activity is usually a prerequisite for proper Golgi localization of furin, the trafficking of the mutant furin proteins was also examined by transfection and immunofluorescence. The 0104S, 01985, and C253S mutants showed normal localization to the Golgi apparatus; the 0196S and C226S mutants localized to the endoplasmic reticulum.

Iodoacetamide Tagging of Critical Cysteine Residues Within the Catalytic Domain of Furin Provides Evidence of Disulfide Bond Location. Jeremy R. Hogg, Gunther P. Alvarado, Kristin M. Hockman, and Joseph F. Sucic, University of Michigan-Flint, Biology Department, Flint, MI 43502-1950; jsucic@umflint.edu

Furin, the major processing enzyme of the secretory pathway, is widely expressed in eucaryotic organisms and in mammalian cell types. Furin plays an extremely important role in mammalian physiology by endoproteolytically activating a number of growth factors, hormones, receptors, and blood proteins. Molecular modeling of the furin catalytic domain suggests the importance of two disulfide bonds critical to active site structure. The model suggests one disulfide linkage occurs between 0104 and 0253, with another between 0226 and either 0196 or 0198. The proposed model could not definitively assign 0196 or 0198 to the bond. These disulfide bonds have not been previously examined experimentally; therefore, conclusive evidence was needed to establish the location of these critical disulfide bonds within the furin catalytic domain. We have generated a soluble, secreted version of furin that displays similar activity to wild type furin. Using conditioned medium as an enzyme source, a purification scheme for furin wa s developed. Subsequent to purification, furin was digested with the endoprotease, Arg C. Fragments of furin containing the aforementioned cysteine residues were isolated and tagged with iodoacetamide, which attaches to the free sulfhydryl group in cysteine residues not involved in a disulfide bond. Analysis of tagged residues resulted in conclusive evidence of disulfide bond location within the furin catalytic domain.

Human Lupus Mouse Model NZM Expresses an Allele with Truncation at the Cytoplasmic Domain of CD72. Sarah Odenbach, Cindy Altes, Masashi Ogata, Sheila Stankevitz, Samantha Miller, Yawei Sun, and Bing Yang, Saginaw Valley State University, Biology Department, 7400 Bay Road, University Center, MI 48710

CD72 is a type II membrane protein expressed in many cell types except T cells and plasma cells. It was suggested to be a negative regulator of B cell activation. The cytoplasmic domain can recruit phosphastase SHP-l that down-regulates the B cell activation signals. Three forms of CD72 were identified in CD72 B allele as the result of alternative mRNA splicing. One form has a 24 amino acid deletion at the cytoplasmic domain. A set of primers that can amplify the complete CD72 cDNA was designed. RT-PCR analysis using this primer set has found that C57BL/6J mice expresses predominantly the full-length mRNA. Lupus susceptible mouse strain NZBWF1 expresses three forms of mRNA with equal density. Homozygous lupus susceptible mouse strain NZM expresses only one form of mRNA. This mRNA has a 72 nucleotide deletion as the result of alternative splicing at the cytoplasmic domain of the protein. Western blotting analysis also confirms that the mRNA expressed in NZM mouse is a form with 72 nucleoride deletion.

Analysis of CD72 Expression in Human Tumor Tissues. Masashi Ogata, Aasia Rehman, Sarah Odenbach, and Bing Yang, Saginaw Valley State University, Biology Department, 7400 Bay Road, University Center, MI 48710

Nearly a quarter of the human population will develop a tumor in a certain stage of our life. It is important to find specific molecular markers for different kinds of tumors and different stages of the tumors. CD72 is a surface protein expressed in many cell types except T cells and plasma cells. It was suggested to be a negative regulator of B cell activation. The function of CD72 in other cell types is unknown. We initiated a study of the function of CD72 in other cell types. In this research, we used western blotting to analyze the CD72 expression in tumor tissues and its surrounding tissues. It was found that among five kidney tumor tissues, one tumor tissue did not express CD72 while the normal surrounding tissue expressed CD72 normally. In tissues from endometrial adenocarcinoma, two out of nine tissues were found to express a CD72 smaller than the normal CD72. Down-regulation and up-regulation of CD72 expression have also been noticed in some of the tumor tissues.

Polymerase Chain Reaction Analysis of CD72 Mutation in Human Tumor Tissues. Kelly Liddle, Sarah Odenbach, Samantha Miller, and Bing Yang, Saginaw Valley State University, Biology Department, 7400 Bay Road, University Center, MI 48710

CD72 is a type II membrane protein expressed in many cell types except T cells and plasma cells. It was suggested to be a negative regulator of B cell activation. The function of CD72 in other cell types is unknown; however, in cDNA from a library prepared from a pool of seven endometrial adenocarcinoma, mutation of CD72 was found. We initiated an investigation into the mutation at the genomic CD72. A primer set that can amplify the region with mutation in cDNA was designed and this set of primers worked well in polymerase chain reaction (PCR). A deletion in this region would result in a shorter PCR product as predicted from the mutation in cDNA. Nine endometrial adenocarcinoma samples have been analyzed. None of them contained the shorter PCR product predicted from the cDNA mutation, which suggests that either the mutation is rare or the mutation is the result of pre-mRNA splicing. When the cDNA from these tumor samples were analyzed by RT-PCR, all the samples including the normal surrounding tissues contain ed unspliced Intron 8. In addition, there are four nucleotides that are deleted in one sample. The significance of this finding is under investigation.
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Publication:Michigan Academician
Geographic Code:1U3MI
Date:Mar 22, 2003
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