ognizant Communication Corporation

Gene Expression, Vol. 9, pp. 157-171
1052-2166/01 $20.00 + .00
Copyright © 2001 Cognizant Comm. Corp.
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¹Division of Environmental Health Sciences, School of Public Health, College of Medicine and Public Health, The Ohio State University, Columbus, OH 43210
²Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus, OH 43210
³Laboratory of Comparative Carcinogenesis, National Cancer Institute-FCRDC, Frederick, MD 21702
⁴Comprehensive Cancer Center, The Ohio State University, Columbus, OH 43210

Transforming growth factor-b (TGF-b) is a potent inhibitor of growth and proliferation of breast epithelial cells, and loss of sensitivity to its effects has been associated with malignant transformation and tumorigenesis. The biological effects of TGF-b are mediated by the TGF-b receptor complex, a multimer composed of TGF-b receptor type I (TbR-I) and TGF-b receptor type II (TbR-II) subunits. Evidence suggests that loss of expression of TbR-II is implicated in the loss of sensitivity of tumorigenic breast cell lines to TGF-b-mediated growth inhibition. A panel of human breast cell lines, including the immortalized MCF-10F and tumorigenic MCF-7, ZR75-1, BT474, T47-D, MDA-MB231, BT20, and SKBR-3 cell lines, was characterized for responsiveness to TGF-b-induced G₁ growth arrest. Only the nontumorigenic MCF-10F and the tumorigenic MDA-MB231 cell lines demonstrated a significant inhibitory response to TGF-b1 and a significant binding of ¹²⁵I-labeled TGF-b ligand. While expression of TbR-I mRNA was similar across the panel of cell lines, TbR-II mRNA expression was decreased significantly in all seven tumorigenic cell lines in comparison with the nontumorigenic MCF-10F cell line. When total cellular protein was fractionated by centrifugation, TbR-I protein was observed in both the cytosolic and membrane fractions at similar levels in all cell lines; however, TbR-II protein was present in the cytosolic fraction in all cell lines, but was observed in the membrane fraction of only the TGF-b-responsive MCF-10F and MDA-MB231 cells. Thus, lack of membrane-bound TbR-II protein appears to be an important determinant of resistance to TGF-b-mediated growth inhibition in this group of breast cell lines.

Key words: Breast cancer; Transforming growth factor-b; Transforming growth factor-b receptors; Growth inhibition

Address correspondence to Christopher M. Weghorst, Ph.D., The Ohio State University, Division of Environmental Health Sciences, Room 1148, CHRI, 300 W. 10th Ave., Columbus, OH 43210-1240. Tel: (614) 293-3713; Fax: (614) 293-3333; E-mail: weghorst.2@osu.edu
 

Departments of ¹Pathology and ²Urology, Northwestern University Medical School, Chicago, IL 60611-3008

Spontaneous peroxisome proliferation-related pleiotropic responses occurring in the liver of mice lacking peroxisomal fatty acyl-CoA oxidase (AOX^-/-) are attributed to sustained activation of peroxisome proliferator-activated receptor a (PPARa) by its putative natural ligands that require AOX for their metabolism. In this study, using a gene expression screen, we show that Ly-6 (lymphocyte antigen 6 complex, locus D; mouse ThB), which belongs to a distinctive family of low molecular weight phosphatidyl inositol anchored cell surface glycoproteins, is upregulated in mouse liver with peroxisome proliferation. Increases in Ly-6D mRNA levels are observed in AOX^-/- mouse liver with spontaneous peroxisome proliferation and also in the liver of wild-type mice treated with synthetic peroxisome proliferators. Peroxisome proliferators failed to increase hepatic Ly-6D mRNA levels in mice lacking PPARa (PPARa^-/-), suggesting a regulatory role for PPARa in the induction of Ly-6D. These observations suggest that changes in certain cell surface proteins also form part of the pleiotropic responses associated with peroxisome proliferation.

Key words: PPARa; Ly-6D; Fatty acyl-CoA oxidase; Peroxisome proliferators

Address correspondence to Janardan K. Reddy, M.D., Department of Pathology, Northwestern University Medical School, 303 East Chicago Avenue, Chicago, IL 60611-3008. Tel: (312) 503-8144; Fax: (312) 503-8249; E-mail: jkreddy@northwestern.edu

¹Department of Urology, ²Medical Scientist Training Program (MSTP), ³Department of Pathology, ⁴Department of Molecular Pharmacology and Biological Chemistry, and ⁵Robert H. Lurie Comprehensive Cancer Center, Northwestern University Medical School, Chicago, IL 60611

The growth and development of some of the male sex accessory organs such as the prostate requires the conversion of testosterone to dihydrotestosterone (DHT) by 5a-reductase. To provide insights into the role of testosterone versus DHT in the prostate, we studied the impact of finasteride, a potent and specific inhibitor of 5a-reductase, on the expression of prostatic androgen-response genes in testis-intact rats and in 7-day castrated rats. Finasteride inhibition of the conversion of testosterone to DHT was confirmed by measuring serum and intraprostatic androgens. As expected, finasteride treatment caused a reduction in the wet weight of the prostate in the testis-intact rats and inhibited the testosterone-stimulated prostatic regrowth in the 7-day castrated rats. Although finasteride treatment had little or no effect on the expression of the surveyed androgen-response genes in testis-intact rats, its administration enhanced the expression of many androgen-response genes during the testosterone-stimulated regrowth of the regressed prostate in castrated rats. These observations suggest that testosterone is more potent than DHT in stimulating the expression of many androgen-response genes in the regressed prostate. The expression of androgen-response genes is mainly prostate specific and thus is likely to be associated with androgen-dependent prostatic differentiation. Therefore, testosterone is more potent than DHT in inducing differentiation and weaker in stimulating proliferation during prostate regrowth. The fact that testosterone is a strong inducer of prostatic differentiation has potential clinical implications.

Key words: 5a-Reductase; Androgen-response genes; Testosterone; Dihydrotestosterone

Address correspondence to Zhou Wang, Department of Urology, Tarry 11-715, Northwestern University Medical School, Chicago, IL 60611. Tel: (312) 908-2264; Fax: (312) 908-7275; E-mail: wangz@northwestern.edu

¹Cell Stress and Apoptosis Research Group, Department of Biochemistry, National University of Ireland, Galway, Ireland
²Institute of Environmental Medicine, Karolinska Institutet, Box 210, S-171 77, Stockholm, Sweden

Mitochondrial cytochrome c release in response to pro-apoptotic signals leads to the formation of a cytochrome c/Apaf-1/procaspase-9 complex (the apoptosome) and resultant activation of caspase-9 and caspase-3. Here we demonstrate that the molecular chaperone, Hsp27, inhibits this cytochrome c-mediated activation of caspase-3. Immunodepeletion of Hsp27 from cytochrome c-activated cytosols resulted in decreased caspase activity. Furthermore, immunoprecipitation of Hsp27 resulted in the coprecipitation of both cytochrome c and procaspase-3. In reciprocal experiments, immunoprecipitation of both procaspase-3 and cytochrome c resulted in coprecipitation of Hsp27, indicating two independent interactions. These results point to Hsp27 mediating its inhibition of procaspase-3 activation through its ability to sequester both cytochrome c and procaspase-3, and thus prevent the correct formation/function of the apoptosome complex.

Key words: Apoptosis; Caspase; Cytochrome c; Hsp27; Stress

Address correspondence to Afshin Samali, Cell Stress and Apoptosis Research Group, Department of Biochemistry, National University of Ireland, Galway, Ireland. Tel: +353-91-750393; Fax: +353-91-525700; E-mail: afshin.samali@nuigalway.ie

¹Department of Molecular and Cellular Biochemistry, College of Medicine, The Ohio State University, 333 Hamilton Hall, 1645 Neil Avenue, Columbus, OH 43210
²Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195

Although the existence of repressor protein(s) involved in the regulation of highly inducible metallothionein-I (MT-I) gene expression has been postulated, none has been identified to date. We considered nuclear factor I (NFI) protein as a potential repressor, as three half-sites for NFI binding are present on MT-I promoter and NFI is known to downregulate several cellular gene promoters. Overexpression of all four isoforms of mouse NFI protein (NFI-A, -B, -C, and -X) suppressed both constitutive and heavy metal-induced activation of the MT-I promoter in HepG2 cells. However, unlike other target genes of NFI, direct interaction of NFI with MT-I promoter is not necessary to mediate its repression. Point mutation of the NFI binding sites within the MT-I promoter that abrogates NFI binding in vitro could not alleviate the repression. Similarly, NFI proteins also repress activity of minimal MT-I promoter deficient in the NFI binding sites. Further, an NFI-C deletion mutant lacking the DNA binding domain continued to repress MT-I promoter. Overexpression of MTF-1, the key trans-acting factor involved in MT-I gene transcription, surmounted NFI-mediated repression of the basal and zinc-induced MT-I promoter activity. These data demonstrate that NFI is a repressor of MT-I expression, where its DNA binding activity is not essential to downregulate the MT-I promoter. Interaction of NFI with another protein(s), probably MTF-1, may be involved in this repression.

Key words: Nuclear factor I (NFI); Metallothionein; Metal transcription factor-1 (MTF-1)

Address correspondence to Samson T. Jacob, Department of Molecular and Cellular Biochemistry, College of Medicine, The Ohio State University, 333 Hamilton Hall, 1645 Neil Avenue, Columbus, OH 43210. Tel: (614) 688-5494; Fax: (614) 688-5600; E-mail: jacob.42@osu.edu

Departments of ¹Molecular Genetics, ²Psychiatry, and ³The Psychiatric Institute, University of Illinois at Chicago, College of Medicine, Chicago, IL 60607

To assess the role of hepatocyte nuclear factor-3b (HNF-3b) in hepatocyte-specific gene transcription, we reported the characterization of the liver phenotype with transgenic mice in which the -3-kb transthyretin (TTR) promoter functioned to increase HNF-3\GK\b expression. During breeding of the TTR-HNF-3b transgenic mice we noticed that they displayed severe ataxia. In this study, we describe the analysis of our transgenic cerebellar phenotype and demonstrate that ectopic expression of HNF-3b disrupted cerebellar morphogenesis and caused reduction in cerebellar size. In postnatal cerebellum, the HNF-3b transgene expression pattern is colocalized to glial fibrillary acidic protein-positive cerebellar astrocytes and Bergmann glial cells. As a result of protracted expression, the transgenic cerebella are impaired in terms of astrocyte dispersal and formation of Bergmann glial cell processes. This caused a disruption in neuronal cell migration to the cortical laminar layers and Purkinje dendritic arbor maturation, thus leading to diminished foliation. Differential hybridization of cDNA arrays was used to identify altered expression of cerebellar genes, which is consistent with the observed defect in transgenic cerebellar morphogenesis and size as well as glial maturation. These include diminished expression of the brain lipid-binding protein, which is required for glial morphological differentiation, and the basic heli-loop-helix NeuroD/Beta2 and homeodomain Engrailed-2 transcription factors, which are required for normal cerebellar morphogenesis and foliation. Undetectable levels of ataxia telangiectasia (ATM), which is required for proper development of the Purkinje dendritic arbor, were found in postnatal transgenic cerebella. Furthermore, the transgenic cerebella displayed levels of insulin-like growth factor binding protein-1 elevated to 22 times greater than those measured for wild-type cerebella, an elevation consistent with the reduction in transgenic cerebellar size.

Key words: Cerebellar expression; Transgenic mice; Winged helix domain; Granule cells; Purkinje cells; Astrocytes; Neuronal migration; Transthyretin promoter; Reeler mice

Address correspondence to Robert H. Costa, Department of Molecular Genetics (M/C 669), University of Illinois at Chicago, College of Medicine, 900 S. Ashland Ave, Rm. 2220 MBRB, Chicago, IL 60607-7170. Tel: (312) 996-0474; Fax: (312) 355-4010; E-mail: RobCosta@uic.edu

*Second and third authors contributed equally to the work.