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1.
Anal Bioanal Chem ; 407(7): 1801-6, 2015 Mar.
Article in English | MEDLINE | ID: mdl-25616703

ABSTRACT

1,1,3,3-Tetramethylguanidine (TMG), methanol and carbon dioxide were investigated as switchable polarity solvents (SPS) in the simultaneous derivatization and extraction of triacylglycerols for the gas chromatographic (GC) characterization of olive oil. Three commercial olive oils were used as test samples. Results of the developed method did not differ statistically from those provided by reference derivatization procedures. The transesterification reaction was carried out under a very mild condition, one step and in situ, and no particular matrix interferences were evidenced. The method represented the first example of the use of a switchable polarity mixture for the preparation of methyl ester derivatives of fatty acids (FAME).

2.
Cancer Res ; 61(18): 6665-8, 2001 Sep 15.
Article in English | MEDLINE | ID: mdl-11559531

ABSTRACT

The transforming growth factor-beta (TGF-beta) receptor complex and its downstream signaling intermediates constitute a tumor suppressor pathway. In many cancers, expression of TGF-beta type II receptor (TbetaR-II) is markedly decreased. In the present study, we show that the hepatocytes isolated from 15-day-old, but not 9-month-old, mice heterozygous for the deletion of the TbetaR-II gene are slightly less sensitive to the growth-inhibitory effect of TGF-beta when compared with wild-type littermates of same age. In addition, the proliferation index of hepatocytes as indicated by bromodeoxyuridine incorporation is mildly increased in the heterozygous mice. These subtle changes in cellular phenotype did not result in either gross or microscopic abnormality of the liver. The treatment of these mice with the chemical carcinogen, diethylnitrosamine, results in a significantly enhanced tumorigenesis in the liver when compared with the wild-type littermates. Our results demonstrate the gene-dosage effect of TbetaR-II and indicate that the reduced expression of TbetaR-II in mice increases susceptibility to tumorigenesis in the liver.


Subject(s)
Cell Transformation, Neoplastic/genetics , Liver Neoplasms, Experimental/genetics , Receptors, Transforming Growth Factor beta/genetics , Animals , Carcinogens , Diethylnitrosamine , Female , Gene Dosage , Genes, cdc/physiology , Genetic Predisposition to Disease , Heterozygote , Liver/drug effects , Liver/metabolism , Liver/physiology , Liver Neoplasms, Experimental/chemically induced , Liver Neoplasms, Experimental/metabolism , Male , Mice , Mice, Inbred C57BL , Phenobarbital/pharmacology , Pregnancy , Protein Serine-Threonine Kinases , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Receptor, Transforming Growth Factor-beta Type II , Receptors, Transforming Growth Factor beta/biosynthesis , Transforming Growth Factor beta/biosynthesis , Transforming Growth Factor beta/genetics , Transforming Growth Factor beta1
3.
Clin Exp Metastasis ; 18(5): 429-38, 2000.
Article in English | MEDLINE | ID: mdl-11467776

ABSTRACT

The effects of the synthetic retinoid N-(4-hydroxyphenyl) retinamide (4-HPR) on prostate cancer metastasis in vivo were evaluated in the mouse prostate reconstitution (MPR) model. MPRs were produced by infection of either heterozygous (+/-) or nullizygous (-/-) p53-mutant fetal prostatic epithelial cells with the recombinant retrovirus Zipras/myc 9. Previous studies have documented that loss of p53 function potentiates metastasis in this model system. MPRs were grafted into homozygous (+/+) p53 male mice, fed a 4-HPR containing diet or a control diet and maintained until the status of tumor progression dictated sacrifice. Under these experimental conditions, treatment with 4-HPR did not have a significant effect on primary tumor wet weight for either p53 +/- or p53 -/- MPRs. For, p53 +/- MPRs the animals fed the 4-HPR diet had a slight improvement in survival and a significant reduction in the number of mesenteric metastases (P = 0.0477, t-test). Notably, in p53 +/- MPRs the incidence of metastasis to lumbar spine and sternum was 92% in the control animals compared to 54% in the 4-HPR treated animals (P = 0.035, chi2-test). In p53 -/- MPRs there was a trend toward a reduction in the number of soft tissue metastases to lung and liver in the 4-HPR group relative to the control diet group and a statistically significant reduction in the incidence of metastasis to bone was demonstrated in that 50% of control animals versus 30% of 4-HPR treated p53 -/- animals harbored bone metastases (P = 0 < 0.05, chi2-test). Cell lines were established from portions of the primary tumor and from selected metastatic deposits in each experimental group. Clonal analysis, by retroviral integration pattern, indicated increased clonal diversity in both the primary tumors and metastasis-derived cell lines from 4-HPR treated animals relative to the control animals. In vitro treatment with 4-HPR did not reveal discriminating differences between cell lines derived from primary tumors and bone metastases or control and treatment groups in regard to growth arrest or apoptotic responses. Overall these studies indicate limited anti-tumor and anti-metastatic activity in this highly aggressive in vivo mouse model of prostate cancer, yet 4-HPR treatment significantly suppressed the development of bone metastases in p53 +/- and p53 -/- MPRs revealing a novel and potentially clinically useful activity of this retinoid.


Subject(s)
Antineoplastic Agents/pharmacology , Bone Neoplasms/drug therapy , Fenretinide/pharmacology , Prostatic Neoplasms/secondary , Animals , Bone Neoplasms/pathology , Cell Division/drug effects , Diet , Disease Models, Animal , Male , Mice , Prostatic Neoplasms/drug therapy , Prostatic Neoplasms/pathology , Survival Rate , Tumor Cells, Cultured , Tumor Suppressor Protein p53/metabolism
4.
Nat Cell Biol ; 1(5): 260-6, 1999 Sep.
Article in English | MEDLINE | ID: mdl-10559937

ABSTRACT

The generation of animals lacking SMAD proteins, which transduce signals from transforming growth factor-beta (TGF-beta), has made it possible to explore the contribution of the SMAD proteins to TGF-beta activity in vivo. Here we report that, in contrast to predictions made on the basis of the ability of exogenous TGF-beta to improve wound healing, Smad3-null (Smad3ex8/ex8) mice paradoxically show accelerated cutaneous wound healing compared with wild-type mice, characterized by an increased rate of re-epithelialization and significantly reduced local infiltration of monocytes. Smad3ex8/ex8 keratinocytes show altered patterns of growth and migration, and Smad3ex8/ex8 monocytes exhibit a selectively blunted chemotactic response to TGF-beta. These data are, to our knowledge, the first to implicate Smad3 in specific pathways of tissue repair and in the modulation of keratinocyte and monocyte function in vivo.


Subject(s)
DNA-Binding Proteins/physiology , Inflammation/physiopathology , Trans-Activators/physiology , Wound Healing/physiology , Wounds and Injuries/physiopathology , Animals , Bone Marrow Cells/cytology , Bone Marrow Cells/physiology , Cell Adhesion , Cell Division , Cells, Cultured , Chemotaxis , DNA-Binding Proteins/deficiency , DNA-Binding Proteins/genetics , Gene Expression Regulation , Inflammation/genetics , Keratinocytes/cytology , Keratinocytes/physiology , Male , Mice , Mice, Knockout , Monocytes/cytology , Monocytes/physiology , Signal Transduction , Skin/injuries , Smad3 Protein , Trans-Activators/deficiency , Trans-Activators/genetics , Transforming Growth Factor beta/genetics , Transforming Growth Factor beta/pharmacology , Transforming Growth Factor beta/physiology , Wound Healing/genetics , Wounds and Injuries/genetics , Wounds and Injuries/pathology
5.
Biochem Biophys Res Commun ; 235(1): 10-4, 1997 Jun 09.
Article in English | MEDLINE | ID: mdl-9196026

ABSTRACT

We report here the entire exon/intron structure of the gene encoding the alpha-subunit of human Rab geranylgeranyl transferase (RABGGTA) gene, which is positioned in a tandem head-to-tail arrangement with the transglutaminase 1 (TGM1) gene, and its polyadenylation signal sequence is located just 2.3 kbp upstream of the capsite of TGM1. Even though TGM1 and RABGGTA have different functions, their close localization raised the question as to whether they are functionally related in the epidermis. To address this question, we have studied the expression of the two genes by RT-PCR in normal human epidermal keratinocytes cultured under various differentiation conditions. While the expression of the TGM1 gene is markedly affected by the calcium concentration of the medium, all trans retinoic acid, vitamin D3, and TPA treatment, the expression of the RABGGTA gene was unaffected by these reagents. Taken together, even though these two genes are unusually closely linked, they are not functionally related in the terminal differentiation program of epidermal keratinocytes.


Subject(s)
Alkyl and Aryl Transferases , Genetic Linkage , Keratinocytes/enzymology , Transferases/genetics , Transglutaminases/genetics , Amino Acid Sequence , Base Sequence , Calcium/pharmacology , Cell Differentiation , Cells, Cultured , Cloning, Molecular , DNA, Complementary/chemistry , Epidermis/enzymology , Exons/genetics , Gene Expression Regulation , Humans , Introns/genetics , Molecular Sequence Data , Polymerase Chain Reaction , RNA, Messenger/genetics , RNA, Messenger/metabolism , Transferases/chemistry , Tretinoin/pharmacology
6.
Endocrinology ; 137(8): 3265-73, 1996 Aug.
Article in English | MEDLINE | ID: mdl-8754749

ABSTRACT

In this study we show that a breast cancer cell line (SKBR3) that expresses no E-cadherin and very low levels of beta-catenin protein and exhibits a poorly adhesive phenotype in Matrigel responds to retinoic acid (RA) by a marked increase in epithelial differentiation. Specifically, treatment of cells with all-trans-RA, 9-cis-RA, or a RA receptor alpha-specific ligand resulted in a large increase in cell-cell adhesive strength and stimulated the formation of fused cell aggregates in Matrigel. A retinoid X receptor-specific ligand was ineffective. Exposure of cells to 9-cis-RA for as little as 4 h was sufficient to maintain the adhesive phenotype for at least 4 days. The effects of 9-cis-RA required protein and RNA synthesis, but were not mediated by factors secreted by stimulated cells or by direct cell contact and did not require serum. These 9-cis-RA-induced morphological effects were completely reversed by growing cells in 50 microM Ca2+, suggesting a mechanism involving a 9-cis-RA-induced increase in Ca(2+)-dependent adhesion. Consistent with this, beta-catenin protein levels were markedly elevated in the 9-cis-RA-treated cells, and beta-catenin became localized to a Triton-insoluble pool at regions of cell-cell contact. No change could be detected in beta-catenin steady state messenger RNA levels, but 9-cis-RA did increase beta-catenin protein stability. Treatment of cells with low calcium medium did not prevent the 9-cis-RA-induced increase in total beta-catenin protein, but did prevent its movement to a Triton-insoluble pool at the cell membrane. Among several kinase inhibitors, only the broad spectrum kinase inhibitor staurosporine and the protein kinase C inhibitor bisindoylmaleimide reversed the morphological changes induced by 9-cis-RA. Like treatment with low calcium medium, these inhibitors did not prevent the 9-cis-RA-induced increase in total beta-catenin protein levels, but completely prevented the movement of beta-catenin to the cell membrane. These results point to a role for beta-catenin and serine kinase activity in mediating the action of 9-cis-RA in epithelial differentiation.


Subject(s)
Breast Neoplasms/metabolism , Cytoskeletal Proteins/metabolism , Trans-Activators , Tretinoin/pharmacology , Breast Neoplasms/pathology , Cadherins/metabolism , Calcium/administration & dosage , Calcium/pharmacology , Cell Adhesion/drug effects , Cell Differentiation , Cell Membrane/metabolism , Culture Media , Cytoskeletal Proteins/drug effects , Drug Stability , Epithelium/drug effects , Epithelium/pathology , Female , Humans , Indoles/pharmacology , Maleimides/pharmacology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/metabolism , RNA, Messenger/metabolism , Staurosporine/pharmacology , Tissue Distribution , Tumor Cells, Cultured/drug effects , beta Catenin
8.
Cancer Res ; 55(23): 5621-7, 1995 Dec 01.
Article in English | MEDLINE | ID: mdl-7585644

ABSTRACT

We evaluated the ability of dietary N-(4-hydroxyphenyl)retinamide; 1 alpha,25-dihydroxy-16-ene-23-yne-26,27-hexafluorocholecalcifero l (Ro24-5531); and tamoxifen to inhibit the development of androgen-promoted carcinomas of the accessory sex organs of male Lobund-Wistar rats. Invasive carcinomas of the seminal vesicle (SV) and anterior prostate (AP) were induced in Lobund-Wistar rats with three different combinations of initiator [N-nitroso-N-methylurea (NMU)] and promoter [testosterone propionate (TP)]: (a) high-dose NMU (30 mg/kg) + high-dose TP (20 mg via implant every 2 months); (b) high-dose NMU + low-dose TP (10 mg implanted every 2 months); or (c) low-dose NMU (15 mg/kg) + low-dose TP. During the period of TP administration, rats were fed a diet supplemented with either N-(4-hydroxyphenyl)retinamide (1 or 2 mmol/kg diet), Ro24-5531 (1.25 or 2.5 nmol/kg diet), tamoxifen (0.5 or 5 mg/kg diet), or vehicle alone. After sacrifice at 8.5 or 11 months, the prostate-seminal vesicle complex from each rat was processed in toto and histologically staged as to the extent of tumor involvement. In animals given low-dose TP, all three agents were significantly effective at reducing the incidence of invasive carcinomas of the SV and, to a lesser degree, the AP. Of the three agents, tamoxifen given in high dose (5 mg/kg) had the strongest activity, reducing the occurrence of invasive SV carcinomas from 72-83% in controls to 6% (P = 0.0001) and the occurrence of invasive AP carcinomas from 50-72% to 18-22% (P < 0.05).


Subject(s)
Anticarcinogenic Agents/therapeutic use , Calcitriol/analogs & derivatives , Neoplasms, Experimental/prevention & control , Neoplasms, Hormone-Dependent/prevention & control , Prostatic Neoplasms/prevention & control , Seminal Vesicles , Tamoxifen/therapeutic use , Androgens , Animals , Calcitriol/therapeutic use , Carcinogens , Drug Screening Assays, Antitumor , Male , Methylnitrosourea , Neoplasms, Experimental/chemically induced , Neoplasms, Experimental/pathology , Neoplasms, Hormone-Dependent/chemically induced , Neoplasms, Hormone-Dependent/pathology , Prostate/drug effects , Prostatic Neoplasms/chemically induced , Prostatic Neoplasms/pathology , Rats , Seminal Vesicles/drug effects , Seminal Vesicles/pathology , Testosterone
9.
Cancer Res ; 54(17): 4614-7, 1994 Sep 01.
Article in English | MEDLINE | ID: mdl-8062253

ABSTRACT

We show that 9-cis-retinoic acid (9cRA) is a potent inhibitor of mammary carcinogenesis induced by N-nitroso-N-methylurea in Sprague-Dawley rats. Rats were first treated with a single dose of N-nitroso-N-methylurea (50 mg/kg body weight) and then fed non-toxic levels of 9cRA (120 or 60 mg/kg of diet). 9cRA was highly effective in reducing tumor incidence, average number of tumors per rat, and average tumor burden, as well as extending tumor latency. The combination of 9cRA with low levels of tamoxifen (TAM; fed at either 1.0 or 0.5 mg/kg of diet) was particularly effective; addition of 9cRA to a TAM regimen doubled the number of animals that were tumor-free at autopsy and significantly diminished tumor number and tumor burden. For suppression of carcinogenesis in vivo, 9cRA was much more potent than all-trans-retinoic acid, both as a single agent or in combination with TAM, although both retinoids had equivalent inhibitory effects on DNA synthesis in cultured human breast cancer cell lines. Both 9cRA and all-trans-retinoic acid induce the expression of the adhesion molecule, E-cadherin, in the SK-BR-3 cell line. We suggest that clinical evaluation of the combination of 9cRA and TAM, either for chemoprevention or for adjuvant therapy, should be considered.


Subject(s)
Mammary Neoplasms, Experimental/prevention & control , Tamoxifen/therapeutic use , Tretinoin/analogs & derivatives , Tretinoin/therapeutic use , Animals , Breast Neoplasms/drug therapy , Drug Screening Assays, Antitumor , Humans , Mammary Neoplasms, Experimental/chemically induced , Methylnitrosourea , Rats , Rats, Sprague-Dawley , Tumor Cells, Cultured
10.
Cancer Res ; 54(13): 3413-21, 1994 Jul 01.
Article in English | MEDLINE | ID: mdl-8012960

ABSTRACT

We have established two new epithelial cell lines (NRP-152, NRP-154), with markedly different properties, from the dorsal-lateral prostate of Lobund/Wistar rats treated with N-methyl-N-nitrosourea and testosterone propionate. NRP-152 cells do not form tumors in athymic mice and retain many of the properties of normal prostatic epithelial cells. They produce prostatic acid phosphatase, have functional androgen receptors, and require the combination of several growth factors in addition to serum for optimal growth. Their growth is stimulated by epidermal growth factor, insulin, dexamethasone, cholera toxin, dihydrotestosterone, and testosterone, and their growth is inhibited by transforming growth factor beta s and retinoic acid. These cells also respond to 1,25-dihydroxyvitamin D3 with an early growth stimulation followed by growth inhibition at later times. In contrast, tumorigenic NRP-154 cells lack detectable androgen receptor mRNA and have less stringent growth factor requirements for optimal growth. Growth of NRP-154 cells is stimulated by dexamethasone and insulin, inhibited by transforming growth factor beta 1, but not significantly altered by epidermal growth factor, cholera toxin, dihydrotestosterone, retinoic acid, or 1 alpha,25-dihydroxyvitamin D3. Our data suggest that the NRP-152 and NRP-154 cell lines are suitable systems for analysis of normal prostate growth and prostatic carcinogenesis.


Subject(s)
Prostate , Prostatic Neoplasms , Acid Phosphatase/analysis , Animals , Calcitriol/pharmacology , Cell Division/drug effects , Cell Line , Dihydrotestosterone/pharmacology , Epithelium , Karyotyping , Male , Methylnitrosourea , Mice , Mice, Nude , Prostate/chemistry , Prostate/drug effects , Prostate/enzymology , Prostate/pathology , Prostatic Neoplasms/chemically induced , Prostatic Neoplasms/chemistry , Prostatic Neoplasms/enzymology , Prostatic Neoplasms/pathology , Rats , Rats, Wistar , Receptors, Androgen/analysis , Transforming Growth Factor beta/pharmacology , Tretinoin/pharmacology , Tumor Cells, Cultured
11.
Cancer Res ; 54(7): 1653-6, 1994 Apr 01.
Article in English | MEDLINE | ID: mdl-8137276

ABSTRACT

We have used the vitamin D analogue, 1 alpha,25-dihydroxy-16-ene-23-yne-26,27-hexafluorocholecalcifero l (Ro24-5531), for inhibition of mammary carcinogenesis induced by N-nitroso-N-methylurea (NMU) in Sprague-Dawley rats. Rats were first treated with a single dose of either 15 or 50 mg/kg body weight NMU and then fed Ro24-5531 (2.5 or 1.25 nmol/kg of diet) for 5-7 months. Ro24-5531 significantly extended tumor latency and lessened tumor incidence as well as tumor number in rats treated with the lower dose of NMU. In rats treated with the higher dose of NMU, Ro24-5531 was fed in combination with tamoxifen; in these experiments, Ro24-5531 significantly enhanced the ability of tamoxifen to reduce total tumor burden, as well as to increase the probability that an animal would be tumor free at the end of the experiment. In vitro, Ro24-5531 was 10-100 times more potent than 1,25-dihydroxyvitamin D3 for inhibition of proliferation of human breast cancer cell lines as well as primary cultures of cells from 2 patients with acute myelogenous leukemia. When fed chronically, Ro24-5531 did not elevate serum calcium in the present studies. We propose the new term, "deltanoids," for the set of molecules composed of vitamin D and its synthetic analogues, in a manner similar to the naming of "retinoids" for the corresponding set of molecules related to vitamin A.


Subject(s)
Adenocarcinoma/prevention & control , Anticarcinogenic Agents/therapeutic use , Calcitriol/analogs & derivatives , Mammary Neoplasms, Experimental/prevention & control , Tamoxifen/therapeutic use , Adenocarcinoma/chemically induced , Adenocarcinoma/pathology , Animals , Anticarcinogenic Agents/administration & dosage , Anticarcinogenic Agents/toxicity , Breast Neoplasms , Calcitriol/administration & dosage , Calcitriol/therapeutic use , Calcitriol/toxicity , Calcium/blood , Cell Division/drug effects , Cell Line , Diet , Female , Humans , Mammary Neoplasms, Experimental/chemically induced , Mammary Neoplasms, Experimental/pathology , Methylnitrosourea , Neoplasm Invasiveness , Rats , Rats, Sprague-Dawley , Tamoxifen/administration & dosage , Tumor Cells, Cultured
12.
Cancer Res ; 54(6): 1440-5, 1994 Mar 15.
Article in English | MEDLINE | ID: mdl-8137245

ABSTRACT

We have developed a grading system for the evaluation of the histogenesis of neoplastic lesions of the prostate and seminal vesicle of the laboratory rat. Prostatic and seminal vesicle carcinomas were induced in Lobund-Wistar rats by initiation with 30 mg/kg N-nitroso-N-methylurea i.v., followed by promotion with 40 mg testosterone propionate implants 1 week later and at 3-month intervals thereafter. Experimental and control groups were sacrificed at various time points between 5 and 11 months after dosing with N-nitroso-N-methylurea in order to visualize progressive stages of carcinogenesis of the dorsolateral prostate, the anterior prostate, and the seminal vesicle. A system of staging was created which allows three different categories (in situ change, invasion, desmoplasia) of tumor development to be ranked progressively in a manner conducive to nonparametric analysis. Each category was then further subdivided to create a total of six stages. This system can be used to evaluate agents which modify tumor induction or suppression. The application of this staging system to the measurement of the effects of the synthetic retinoid, 4-hydroxyphenyl retinamide, on prostatic carcinogenesis in the Lobund-Wistar rat is described.


Subject(s)
Genital Neoplasms, Male/pathology , Prostatic Neoplasms/pathology , Seminal Vesicles/pathology , Animals , Evaluation Studies as Topic , Fenretinide/therapeutic use , Genital Neoplasms, Male/prevention & control , Male , Methylnitrosourea , Neoplasm Staging/methods , Prostatic Neoplasms/chemically induced , Prostatic Neoplasms/prevention & control , Rats , Rats, Wistar , Testosterone
13.
Cancer Res ; 53(19): 4461-5, 1993 Oct 01.
Article in English | MEDLINE | ID: mdl-8402613

ABSTRACT

Several epidemiological studies have implicated low dietary and serum levels of retinol with an increased risk for the development of human prostate cancer. In a recent report, dietary fenretinide [N-[(4-hydroxyphenyl)] retinamide], a synthetic retinoid with low toxicity, decreased the incidence of experimentally induced prostate cancer. Fenretinide is currently being evaluated in phase I and phase II clinical trials as an agent for both the treatment and chemoprevention of human prostate cancer. Because of these findings, we investigated whether dietary fenretinide could alter the incidence of phenotype of oncogene-induced prostate cancer in the mouse prostate reconstitution model system. When compared to control-fed animals, dietary fenretinide reduced the tumor incidence by 49% and the tumor mass by 52% of ras+myc-induced cancers in the mouse prostate reconstitution model system, which was modified to prolong the latency period before cancer development. Retinoids have a wide ranging effect on cellular differentiation, growth factor synthesis, and immune function. While its mechanism of action in this system remains unclear, fenretinide is an effective agent for the chemoprevention and growth modulation of oncogene-induced prostate cancer in the mouse prostate reconstitution model system and may be effective for the chemoprevention of human prostate cancer.


Subject(s)
Anticarcinogenic Agents/therapeutic use , Cell Transformation, Neoplastic/drug effects , Fenretinide/therapeutic use , Genes, myc , Genes, ras , Prostate/pathology , Prostatic Neoplasms/prevention & control , Animals , Anticarcinogenic Agents/administration & dosage , Anticarcinogenic Agents/pharmacology , Diet , Fenretinide/administration & dosage , Fenretinide/pharmacology , Fetus , Genetic Vectors , Male , Mice , Mice, Inbred C57BL , Promoter Regions, Genetic , Prostate/drug effects , Prostatic Neoplasms/genetics , Prostatic Neoplasms/pathology , Transfection
14.
Cancer Res ; 53(7): 1480-3, 1993 Apr 01.
Article in English | MEDLINE | ID: mdl-8453611

ABSTRACT

To understand the molecular mechanism of carcinogenesis in androgen-dependent tumors, we have searched for new markers which are associated with this process. In normal rat prostate and seminal vesicle, sulfated glycoprotein 2 (SGP-2) messenger RNA is barely detectable. However, we have found high levels of SGP-2 expression in the epithelial component of carcinomas of the prostate and seminal vesicle after initiation with N-nitroso-N-methylurea and promotion with testosterone propionate. We have also observed induction of SGP-2 expression in epithelial cells at early stages in carcinogenesis when cytologically malignant cells first begin to appear. SGP-2 has been reported previously to be associated with a variety of models of programmed cell death (apoptosis), including the prostate following castration. Our present findings provide a novel marker for carcinogenesis in the rat prostate and seminal vesicle.


Subject(s)
Biomarkers, Tumor/metabolism , Glycoproteins/metabolism , Molecular Chaperones , Prostatic Neoplasms/metabolism , Seminal Vesicles/metabolism , Animals , Blotting, Northern , Clusterin , Epithelium/metabolism , Male , Methylnitrosourea , Prostatic Neoplasms/chemically induced , Prostatic Neoplasms/pathology , Rats
15.
Biochem J ; 283 ( Pt 1): 91-8, 1992 Apr 01.
Article in English | MEDLINE | ID: mdl-1567383

ABSTRACT

Site-directed mutants of transforming growth factor-alpha (TGF-alpha) were expressed in an Escherichia coli outer membrane protein A (ompA) expression/secretion vector under the transcriptional control of the lambda PL promoter. TGF-alpha mutant proteins were isolated from cell pellets using alkaline extraction with 0.1 M-Tris (pH 10.5). The levels of protein expression of 23 TGF-alpha mutants were comparable with those of wild-type TGF-alpha, as determined by immunoblotting and radioimmunoassay. An analysis of biological activity using as assays radioreceptor binding competition and colony formation in soft agar showed that the following mutations destroy the activity of TGF-alpha: Gly-19 to Val, Val-33 to Pro and Gly-40 to Val. Mutations of Arg-42 to Lys, Leu-48 to Ala, Tyr-38 to Trp or Phe-17 to Tyr significantly decrease, but do not destroy, biological activity when compared with the wild-type. Mutations in 14 other residues did not significantly alter receptor binding or colony-forming activity. These studies suggest that two domains localized at the surface of TGF-alpha are important in receptor binding and colony-forming activity. Domain I involves amino acid residues which include Tyr-38 and Leu-48; domain II includes residues Phe-15, Phe-17 and Arg-42.


Subject(s)
Mutagenesis, Site-Directed/genetics , Transforming Growth Factor alpha/genetics , Amino Acid Sequence , Gene Expression/genetics , Humans , Macromolecular Substances , Molecular Sequence Data , Mutation , Protein Conformation , Sequence Homology, Nucleic Acid , Structure-Activity Relationship , Transforming Growth Factor alpha/physiology
16.
Oncol Res ; 4(4-5): 193-200, 1992.
Article in English | MEDLINE | ID: mdl-1324032

ABSTRACT

Over 4,500 natural product extracts were screened for their abilities to inhibit binding of radiolabeled TGF-alpha to A431 cells; several plant extracts were identified as potential leads with IC50 values of less than 30 micrograms/mL. The active components of one extract were purified to homogeneity and identified as the porphyrin structures, methyl pheophorbides a and b. These compounds inhibited both TGF-alpha receptor binding and the TGF-alpha induced proliferation of NRK-49F cells in soft agar. To construct a structure-function relationship, a series of commercially available porphyrin derivatives was evaluated. The most potent compound, hematoporphyrin IX, inhibited TGF-alpha functions in a dose-dependent fashion with IC50 values slightly lower than the methyl pheophorbides. Further studies revealed that inhibition of TGF-alpha binding was light dependent and that inhibition did not involve direct competition of porphyrins for the TGF-alpha binding site. To determine the specificity of inhibition, the porphyrins were tested in a number of other receptor-ligand assays. TNF-alpha and beta-adrenoceptor bindings were unaffected, whereas IL-1 beta binding to EL-4 membranes and platelet-derived growth factor induced thymidine incorporation in NIH-3T3 cells were both antagonized by the most active porphyrins. Inhibition of TGF-beta binding to NRK-49F cells and TGF-beta-induced growth of AKR-2B cells was also observed. In summary, we report that methyl pheophorbides are naturally occurring, photodynamic antagonists of TGF-alpha, and although the inhibitory properties of these molecules were not confined to TGF-alpha alone, some level of receptor selectivity was observed.


Subject(s)
Cell Division/drug effects , Chlorophyll/analogs & derivatives , Cytokines/pharmacology , ErbB Receptors/metabolism , Plant Extracts/pharmacology , Porphyrins/pharmacology , Tissue Extracts/pharmacology , Transforming Growth Factor alpha/metabolism , Transforming Growth Factor alpha/pharmacology , Animals , Cell Line , Chlorophyll/pharmacology , Drug Synergism , ErbB Receptors/antagonists & inhibitors , Humans , Receptors, Adrenergic, beta/metabolism , Recombinant Proteins/metabolism , Recombinant Proteins/pharmacology , Transforming Growth Factor beta/pharmacology
17.
J Cell Biochem ; 44(4): 229-39, 1990 Dec.
Article in English | MEDLINE | ID: mdl-2095367

ABSTRACT

A polypeptide growth factor has been partially purified from medium conditioned by the human adrenocortical carcinoma cell line SW13. This factor, designated h-TGFe, stimulates anchorage-independent growth of the SW13 cells. Similar activity was observed in human milk, and in conditioned media from seven of 14 epithelial cell lines. The SW13-derived activity is stable to low pH and 8M urea but labile to dithiothreitol and 2% sodium dodecyl sulfate. Human TGFe does not bind to heparin and fails to stimulate growth of endothelial cells in monolayer culture. The apparent molecular weight of h-TGFe is 59k by size exclusion chromatography in the presence of 8M urea and the activity binds strongly to cation exchangers. The activity elutes at 15-30% acetonitrile from a C18 reverse-phase column and has been partially purified by using a four-step chromatographic procedure. TGFe appears to be a novel growth factor produced by many epithelial cells and tissues.


Subject(s)
Transforming Growth Factors/isolation & purification , Cell Line , Chromatography, High Pressure Liquid , Dithiothreitol/pharmacology , Dose-Response Relationship, Drug , Epithelial Cells , Epithelium/metabolism , Fibroblast Growth Factors/analysis , Fibroblast Growth Factors/pharmacology , Humans , Hydrogen-Ion Concentration , Milk, Human/chemistry , Molecular Weight , Sodium Dodecyl Sulfate/pharmacology , Transforming Growth Factors/analysis , Transforming Growth Factors/pharmacology , Tumor Cells, Cultured
18.
J Biol Response Mod ; 9(4): 361-7, 1990 Aug.
Article in English | MEDLINE | ID: mdl-2168471

ABSTRACT

The guanine nucleotide binding protein (G-protein) dependency of several of the activities of tumor necrosis factor (TNF), including cytotoxicity, inhibition of lipoprotein lipase activity, blockade of 3T3-L1 differentiation, and receptor binding were examined. TNF induced killing of the TNF-sensitive cell line L929S (ED50 = 30 pM), but had little to no effect on the TNF-resistant cell line L929R (ED50 = 5,300 pM). TNF-induced cytotoxicity in L929S was antagonized in a dose-dependent manner by pertussis toxin (sevenfold increase in ED50). However, TNF-induced cytotoxicity in L929R cells was only minimally affected by pretreatment with a high dose (50 ng/ml) of pertussis toxin (1.5-fold increase in ED50). Parallel biochemical investigations revealed that inhibition was accompanied by toxin-induced ADP ribosylation of a Gi alpha-like subunit in L929 and 3T3-L1 cell membranes. Pertussis toxin also significantly reduced TNF-induced inhibition of lipoprotein lipase activity in 3T3-L1 adipocytes and TNF blockade of 3T3-L1 preadipocyte differentiation. However, pertussis toxin pretreatment of L929S, L929R, and 3T3-L1 cell cultures had little to no effect on TNF receptor binding. These data indicate that several TNF-induced biological activities in the L929 and 3T3-L1 cell lines are partially dependent upon a pertussis toxin-sensitive G-protein.


Subject(s)
GTP-Binding Proteins/physiology , Tumor Necrosis Factor-alpha/physiology , Adenosine Diphosphate Ribose/metabolism , Adipose Tissue/cytology , Adipose Tissue/drug effects , Animals , Cell Differentiation/drug effects , Cell Line , GTP-Binding Proteins/drug effects , GTP-Binding Proteins/metabolism , L Cells/drug effects , Lipoprotein Lipase/antagonists & inhibitors , Pertussis Toxin , Protein Binding/drug effects , Receptors, Cell Surface/metabolism , Receptors, Tumor Necrosis Factor , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Tumor Necrosis Factor-alpha/metabolism , Virulence Factors, Bordetella
19.
Int J Cancer ; 44(1): 110-5, 1989 Jul 15.
Article in English | MEDLINE | ID: mdl-2545633

ABSTRACT

Previous studies have established that colon carcinoma cells secrete several polypeptide growth factors, including TGF-alpha/EGF and TGF-beta, suggesting that these and related molecules function in an autocrine/paracrine fashion to modulate tumor-cell growth. To investigate this possibility, we have studied the expression of transforming growth factor receptors in a panel of human colon carcinoma cell lines and in several untransformed epithelial cell populations. The results have revealed that neoplastic colon cells express receptors for both TGF-alpha/EGF and TGF-beta. Immunoprecipitation identified the TGF-alpha/EGF receptor as a structurally intact 170-kDa protein. No evidence for over-expression was found. TGF-alpha (and EGF) enhanced receptor autophosphorylation, indicating that these receptors were biochemically functional. TGF-beta blocked DNA synthesis in non-neoplastic epithelial cells but not in tumorigenic colon populations. There was no correlation with TGF-beta receptor number or dissociation constant. However, chemical cross-linking studies revealed a TGF-beta receptor subtype of 75 kDa in 3 of the 4 colon carcinoma cells which was undetectable in normal IEC epithelial cultures, suggesting a possible association between 75-kDa receptor expression and refractoriness to growth inhibition of TGF-beta. Together, these data support the concept that locally-produced growth regulators can function in an autocrine or paracrine manner to influence the proliferation of colon carcinoma cells.


Subject(s)
Carcinoma/analysis , Colonic Neoplasms/analysis , Epidermal Growth Factor/analysis , ErbB Receptors/analysis , Receptors, Cell Surface/analysis , Transforming Growth Factors/metabolism , Carcinoma/pathology , Colonic Neoplasms/pathology , Humans , Receptors, Transforming Growth Factor beta , Tumor Cells, Cultured
20.
Cancer Res ; 49(11): 2898-904, 1989 Jun 01.
Article in English | MEDLINE | ID: mdl-2541895

ABSTRACT

Conditioned media collected under serum-free conditions over 24 to 48 h from 18 human colon adenocarcinoma cell lines were analyzed for transforming growth factor, types alpha and beta (TGF-alpha and -beta), and platelet-derived growth factor in assays for anchorage-independent growth and radioreceptor competition. Detectable levels of TGF-alpha, TGF-beta, and platelet-derived growth factor were produced by 17, 16, and 6 cell lines, respectively. Three liters of conditioned medium from highly tumorigenic (HT-29, DLD-1, and SW620) and nontumorigenic (SKCO-1) colon cell lines and from nonneoplastic rat kidney (NRK-52E) and small intestinal (IEC-6) epithelial cells were purified by high-performance liquid chromatography and assayed for TGF-alpha- and TGF-beta-like activity. The highly tumorigenic colon cell lines produced 10- to 45-fold (soft agar), 19- to 90-fold (radioreceptor), and 4- to 35-fold (radioimmunoassay) more TGF-alpha activity compared to the nonneoplastic rat intestinal (IEC) epithelial cells. NRK-52E did not produce detectable TGF-alpha activity. Radioimmunoassay analysis of peak fractions revealed only TGF-alpha immunoreactivity; epidermal growth factor was not detected. Levels of TGF-beta-like material in the colon carcinoma populations were comparable (HT-29) or elevated (DLD-1, SW620) only 3- to 4-fold (soft agar) or 1- to 3-fold (radioreceptor binding) compared to IEC cells or NRK-52E. Growth factor production is an ubiquitous property of colon carcinoma cell lines maintained in vitro and is consistent with this class of molecule, playing a contributory role in regulating cell growth.


Subject(s)
Colonic Neoplasms/metabolism , Colorectal Neoplasms/metabolism , ErbB Receptors/biosynthesis , Platelet-Derived Growth Factor/biosynthesis , Receptors, Cell Surface/biosynthesis , Transforming Growth Factors/biosynthesis , Animals , Chromatography, High Pressure Liquid , Culture Media/analysis , Dose-Response Relationship, Drug , Humans , Rats , Receptors, Transforming Growth Factor beta , Tumor Cells, Cultured/metabolism
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