Thrombospondin-1 Gene Expression Affects Survival and Tumor Spectrum of p53-Deficient Mice
2001; Elsevier BV; Volume: 159; Issue: 5 Linguagem: Inglês
10.1016/s0002-9440(10)63042-8
ISSN1525-2191
AutoresJack Lawler, Weimin Miao, Mark Duquette, Noël Bouck, Roderick T. Bronson, Richard O. Hynes,
Tópico(s)Lymphatic System and Diseases
ResumoIn vitro and in vivo data indicate that thrombospondin-1 (TSP1) inhibits tumor progression in several ways including direct effects on cellular growth and apoptosis in the stromal compartment. To evaluate the importance of TSP1 for the progression of naturally arising tumors in vivo, we have crossed TSP1-deficient mice with p53-deficient mice. In p53-null mice, the absence of TSP1 decreases survival from 160 ± 52 days to 149 ± 42 days. A log-rank test comparing survival curves for these two populations yields a two-sided P value of 0.0272. For mice that are heterozygous for the p53-null allele, survival is 500 ± 103 days in the presence of TSP1 expression, and 426 ± 125 days in its absence (P = 0.0058). Whereas TSP1 expression did not cause a measurable change in the incidence of the majority of tumor types, a statistically significant (P ≤ 0.05) decrease in the incidence of osteosarcomas is observed in the absence of TSP1. To determine more directly if host TSP1 inhibits tumor growth, B16F10 melanoma and F9 testicular teratocarcinoma cells have been implanted in C57BL/6J and 129Sv TSP1-null mice, respectively. The B16F10 tumors grow approximately twice as fast in the TSP1-null background and exhibit an increase in vascular density, a decrease in the rate of tumor cell apoptosis, and an increase in the rate of tumor cell proliferation. Increased tumor growth is also observed in the absence of TSP1 on the 129Sv genetic background. These data indicate that endogenous host TSP1 functions as a modifier or landscaper gene to suppress tumor growth. In vitro and in vivo data indicate that thrombospondin-1 (TSP1) inhibits tumor progression in several ways including direct effects on cellular growth and apoptosis in the stromal compartment. To evaluate the importance of TSP1 for the progression of naturally arising tumors in vivo, we have crossed TSP1-deficient mice with p53-deficient mice. In p53-null mice, the absence of TSP1 decreases survival from 160 ± 52 days to 149 ± 42 days. A log-rank test comparing survival curves for these two populations yields a two-sided P value of 0.0272. For mice that are heterozygous for the p53-null allele, survival is 500 ± 103 days in the presence of TSP1 expression, and 426 ± 125 days in its absence (P = 0.0058). Whereas TSP1 expression did not cause a measurable change in the incidence of the majority of tumor types, a statistically significant (P ≤ 0.05) decrease in the incidence of osteosarcomas is observed in the absence of TSP1. To determine more directly if host TSP1 inhibits tumor growth, B16F10 melanoma and F9 testicular teratocarcinoma cells have been implanted in C57BL/6J and 129Sv TSP1-null mice, respectively. The B16F10 tumors grow approximately twice as fast in the TSP1-null background and exhibit an increase in vascular density, a decrease in the rate of tumor cell apoptosis, and an increase in the rate of tumor cell proliferation. Increased tumor growth is also observed in the absence of TSP1 on the 129Sv genetic background. These data indicate that endogenous host TSP1 functions as a modifier or landscaper gene to suppress tumor growth. The p53 tumor suppressor gene product is a transcription factor that induces growth arrest and apoptosis.1Giaccia AJ Kastan MB The complexity of p53 modulation: emerging patterns from divergent signals.Genes Dev. 1998; 12: 2973-2983Crossref PubMed Scopus (1174) Google Scholar Mutations in the p53 gene are common in human cancers. In the mouse, the complete absence of p53 expression results in a dramatic decrease in survival, with all of the mice succumbing to various types of cancer within 9 months.2Donehower LA Harvey M Slagle BL McArthur MJ Montgomery Jr, CA Butel JS Bradley A Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumors.Nature. 1992; 356: 215-221Crossref PubMed Scopus (4020) Google Scholar, 3Jacks T Remington L Williams BO Schmitt EM Halachmi S Bronson RT Weinberg RA Tumor spectrum analysis in p53-mutant mice.Curr Biol. 1994; 4: 1-7Abstract Full Text Full Text PDF PubMed Scopus (1732) Google Scholar, 4Harvey M McArthur MJ Montgomery Jr, C Bradley A Donehower LA Genetic background alters the spectrum of tumors that develop in p53-deficient mice.FASEB J. 1993; 7: 938-943Crossref PubMed Scopus (236) Google Scholar Lymphomas are observed with the highest frequency in these mice; however, tumors from a wide range of cell lineages are observed. Mice that are heterozygous for p53 have an increased life span but eventually succumb to a more evenly distributed range of lymphomas, sarcomas, and carcinomas. Thus, mice that are deficient in p53 gene expression are a valuable model for assaying the effects of other gene mutations on the progression of naturally occurring tumors. The loss of p53 function in fibroblasts from patients with Li-Fraumeni Syndrome has been shown to correlate with a reduction in thrombospondin-1 (TSP1) protein expression and a switch in the angiogenic phenotype from inhibitory to stimulatory.5Stellmach V Volpert OV Crawford SE Lawler J Hynes RO Bouck N Tumour suppressor genes and angiogenesis: the role of TP53 in fibroblasts.Eur J Cancer. 1996; 32A: 2394-2400Abstract Full Text PDF PubMed Scopus (37) Google Scholar, 6Volpert OV Damern KM Bouck N Sequential development of an angiogenic phenotype by human fibroblasts progressing to tumorigenicity.Oncogene. 1997; 14: 1495-1502Crossref PubMed Scopus (176) Google Scholar Moreover, p53 can also regulate TSP1 and angiogenesis in cultured fibroblasts and in some human breast tumors.7Volpert OV Stellmach V Bouck N The modulation of thrombospondin and other naturally occurring inhibitors of angiogenesis during tumor progression.Breast Cancer Res Treat. 1995; 36: 119-126Crossref PubMed Scopus (95) Google Scholar However, in other tissues, such as brain, skin, and bladder, p53 does not seem to regulate TSP1 expression.8Hsu SC Volpert OV Steck PA Mikkelsen T Polverini PJ Rao S Chou P Bouck NP Inhibition of angiogenesis in human glioblastomas by chromosome 10 induction of thrombospondin-1.Cancer Res. 1996; 56: 5684-5691PubMed Google Scholar, 9Wang XJ Greenhalgh DA Jiang A He D Zhong L Brinkley BR Roop DR Analysis of centrosome abnormalities and angiogenesis in epidermal-targeted p53172H mutant and p53-knockout mice after chemical carcinogenesis: evidence for a gain of function.Mol Carcinog. 1998; 23: 185-192Crossref PubMed Scopus (52) Google Scholar, 10Campbell SC Volpert OV Ivanovich M Bouck NP Molecular mediators of angiogenesis in bladder cancer.Cancer Res. 1998; 58: 1298-1304PubMed Google Scholar The thrombospondins are a family of extracellular calcium-binding proteins.11Adams JC Tucker RP Lawler J The Thrombospondin Gene Family. R.G. Landes Co., Austin1995Google Scholar, 12Bornstein P Thrombospondins: structure and regulation of expression.FASEB J. 1992; 6: 3290-3299Crossref PubMed Scopus (306) Google Scholar, 13Roberts DD Regulation of tumor growth and metastasis by thrombospondin-1.FASEB J. 1996; 10: 1183-1191Crossref PubMed Scopus (243) Google Scholar Of the five family members, TSP1 is the most extensively characterized because it is readily purified from blood platelets. Through its interaction with proteoglycans, other matrix proteins, growth factors, and membrane receptors, TSP1 directs the assembly of multiprotein complexes that modulate cellular phenotype. TSP1 also directly activates transforming growth factor-β (TGF-β) and can affect the activity of various extracellular proteases including plasmin, elastase, and cathepsin.14Schultz-Cherry S Chen H Mosher DF Misenheimer TM Krutzsch HC Roberts DD Murphy-Ullrich JE Regulation of transforming growth factor-beta activation by discrete sequences of thrombospondin 1.J Biol Chem. 1995; 270: 7304-7310Crossref PubMed Scopus (372) Google Scholar, 15Crawford SE Stellmach V Murphy-Ullrich JE Ribeiro SMF Lawler J Hynes RO Boivin GP Bouck N Thrombospondin-1 is a major activator of TGF-β1 in vivo.Cell. 1998; 93: 1159-1170Abstract Full Text Full Text PDF PubMed Scopus (982) Google Scholar, 16Mosher DF Misenheimer TM Stenflo J Hogg PJ Modulation of fibrinolysis by thrombospondin.Ann NY Acad Sci. 1992; 667: 64-69Crossref PubMed Scopus (21) Google Scholar, 17Anonick PK Yoo JK Webb DJ Gonias SL Characterization of the antiplasmin activity of human thrombospondin-1 in solution.Biochem J. 1993; 289: 903-909PubMed Google Scholar, 18Hogg PJ Jiménez BM Chesterman CN Identification of possible inhibitory reactive centers in thrombospondin 1 that may bind cathepsin G and neutrophil elastase.Biochemistry. 1994; 33: 6531-6537Crossref PubMed Scopus (25) Google Scholar Biological processes frequently involve a balance of stimulatory and inhibitory factors.19Bouck N Stellmach V Hsu SC How tumors become angiogenic.Adv Cancer Res. 1996; 69: 135-174Crossref PubMed Google Scholar, 20Hanahan D Folkman J Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis.Cell. 1996; 86: 353-364Abstract Full Text Full Text PDF PubMed Scopus (6036) Google Scholar, 21Dawson DW Bouck NP Thrombospondin as an inhibitor of angiogenesis.in: Teicher BA Antiangiogenic Agents in Cancer Therapy. Humana Press Inc., Totowa1999: 185-203Crossref Google Scholar The multiprotein complexes that are formed on the cell membrane in response to TSP1 modulate cellular phenotype by shifting these balances. The resulting changes in extracellular protease or angiogenic activity are important at sites of tissue remodeling during wound healing and tumor progression. TSP1 supports attachment and migration of various carcinoma and melanoma cell lines.22Guo N-H Krutzsch HC Negre E Vogel T Blake DA Roberts DD Heparin- and sulfatide-binding peptides from the type 1 repeats of human thrombospondin promote melanoma cell adhesion.Proc Natl Acad Sci USA. 1992; 89: 3040-3044Crossref PubMed Scopus (141) Google Scholar, 23Guo N-H Zabrenetzky VS Chandrasekaran L Sipes JM Lawler J Krutzsch HC Roberts DD Differential roles of protein kinase C and pertussis toxin-sensitive G-proteins in modulation of melanoma cell proliferation and motility by thrombospondin-1.Cancer Res. 1998; 58: 3154-3162PubMed Google Scholar, 24Taraboletti G Roberts DD Liotta LA Thrombospondin-induced tumor cell migration: heptotaxis and chemotaxis are mediated by different molecular domains.J Cell Biol. 1987; 105: 2409-2415Crossref PubMed Scopus (174) Google Scholar In addition, TSP1 inhibits proliferation of endothelial cells in vitro and inhibits angiogenesis in vivo.25Good DJ Polverini PJ Rastinejad F LeBeau MM Lemons RS Frazier WA Bouck NP A tumor suppressor-dependent inhibitor of angiogenesis is immunologically and functionally indistinguishable from a fragment of thrombospondin.Science. 1990; 87: 6624-6628Google Scholar These effects are mediated by various receptors on the cell surface, including proteoglycans, integrins, integrin-associated protein (IAP), CD36, and several receptors that remain to be fully characterized.11Adams JC Tucker RP Lawler J The Thrombospondin Gene Family. R.G. Landes Co., Austin1995Google Scholar A 50,000-d protein that is expressed in tumor tissue is included in the latter group.26Tuszynski GP Rothman VL Papale M Hamilton BK Eyal J Identification and characterization of a tumor cell receptor for CSVTCG, a thrombospondin adhesive domain.J Cell Biol. 1993; 120: 513-521Crossref PubMed Scopus (72) Google Scholar This receptor, CD36, and some proteoglycans appear to bind to the type 1 repeats of TSP1. Fusion proteins or peptides that contain type 1 repeat sequences inhibit angiogenesis, inhibit proliferation of melanoma cells, and inhibit tumor growth.23Guo N-H Zabrenetzky VS Chandrasekaran L Sipes JM Lawler J Krutzsch HC Roberts DD Differential roles of protein kinase C and pertussis toxin-sensitive G-proteins in modulation of melanoma cell proliferation and motility by thrombospondin-1.Cancer Res. 1998; 58: 3154-3162PubMed Google Scholar, 27Tolsma SS Volpert OV Good DJ Frazier WA Polverini PJ Bouck N Peptides derived from two separate domains of the matrix protein thrombospondin-1 have anti-angiogenic activity.J Cell Biol. 1993; 122: 497-511Crossref PubMed Scopus (509) Google Scholar, 28Guo N-H Drutzch HC Inman JK Roberts DD Thrombospondin 1 and type 1 repeat peptides of thrombospondin 1 specifically induce apoptosis of endothelial cells.Cancer Res. 1997; 57: 1735-1742PubMed Google Scholar, 29Guo N-H Krutzsch HC Inman JK Shannon CS Roberts DD Antiproliferative and antitumor activities of D-reversed peptides derived from the second type 1 repeat of thrombospondin-1.J Peptide Res. 1997; 50: 210-221Crossref PubMed Scopus (63) Google Scholar In addition, a peptide from the procollagen homology region has been shown to inhibit angiogenesis.27Tolsma SS Volpert OV Good DJ Frazier WA Polverini PJ Bouck N Peptides derived from two separate domains of the matrix protein thrombospondin-1 have anti-angiogenic activity.J Cell Biol. 1993; 122: 497-511Crossref PubMed Scopus (509) Google Scholar Inhibition of angiogenesis by TSP1 is mediated by CD36 on endothelial cells.30Dawson DW Pearce SFA Zhong R Silverstein RL Frazier WA Bouck NP CD36 mediates the in vitro inhibitory effects of thrombospondin-1 on endothelial cells.J Cell Biol. 1997; 138: 707-717Crossref PubMed Scopus (537) Google Scholar, 31Jimenez B Volpert OV Crawford SE Febbraio M Silverstein RL Bouck N Signals leading to apoptosis-dependent inhibition of neovascularization by thrombospondin-1.Nat Med. 2000; 6: 41-48Crossref PubMed Scopus (845) Google Scholar Recent data indicate that CD36 associates with integrins and tetraspanins on the platelet membrane.32Miao W Seng WL Duquette M Laus C Detmar M Lawler J Thrombospondin-1 type 1 repeat recombinant proteins inhibit tumor growth.Blood. 2001; 97: 1689-1696Crossref PubMed Scopus (77) Google Scholar TSP1 is expressed in normal breast, colon, and bladder epithelium.10Campbell SC Volpert OV Ivanovich M Bouck NP Molecular mediators of angiogenesis in bladder cancer.Cancer Res. 1998; 58: 1298-1304PubMed Google Scholar, 33Bertin N Clezardin P Kubiak R Frappart L Thrombospondin-1 and -2 messenger RNA expression in normal, benign and neoplastic human breast tissues: correlation with prognostic factors, tumor angiogenesis and fibroblastic desmoplasia.Cancer Res. 1997; 57: 396-399PubMed Google Scholar, 34Grossfeld GD Ginsberg DA Stein JP Bochner BH Esrig D Grosher S Dunn M Nichols PW Taylor CR Skinner DG Cote RJ Thrombospondin-1 expression in bladder cancer: association with p53 alterations, tumor angiogenesis and tumor progression.J Natl Cancer Inst. 1997; 89: 219-227Crossref PubMed Scopus (296) Google Scholar, 35Tokunaga T Nakamura M Oshika Y Tsuchida T Kazuno M Fukushima Y Kawai K Abe Y Kijima H Yamazaki H Tamaoki N Ueyama Y Alterations in tumour suppressor gene p53 correlate with inhibition of thrombospondin-1 gene expression in colon cancer cells.Virchows Arch. 1998; 433: 415-418Crossref PubMed Scopus (36) Google Scholar In general, TSP1 expression is significantly reduced in cancer cells that arise in these tissues. However, lobular carcinomas in the breast express significantly higher levels of TSP1 and TSP2 than normal tissue.10Campbell SC Volpert OV Ivanovich M Bouck NP Molecular mediators of angiogenesis in bladder cancer.Cancer Res. 1998; 58: 1298-1304PubMed Google Scholar, 33Bertin N Clezardin P Kubiak R Frappart L Thrombospondin-1 and -2 messenger RNA expression in normal, benign and neoplastic human breast tissues: correlation with prognostic factors, tumor angiogenesis and fibroblastic desmoplasia.Cancer Res. 1997; 57: 396-399PubMed Google Scholar, 34Grossfeld GD Ginsberg DA Stein JP Bochner BH Esrig D Grosher S Dunn M Nichols PW Taylor CR Skinner DG Cote RJ Thrombospondin-1 expression in bladder cancer: association with p53 alterations, tumor angiogenesis and tumor progression.J Natl Cancer Inst. 1997; 89: 219-227Crossref PubMed Scopus (296) Google Scholar, 35Tokunaga T Nakamura M Oshika Y Tsuchida T Kazuno M Fukushima Y Kawai K Abe Y Kijima H Yamazaki H Tamaoki N Ueyama Y Alterations in tumour suppressor gene p53 correlate with inhibition of thrombospondin-1 gene expression in colon cancer cells.Virchows Arch. 1998; 433: 415-418Crossref PubMed Scopus (36) Google Scholar TSP1 expression shows an inverse correlation with vascular density in bladder and colon cancer but does not correlate with vascular density in some other tissues such as ductal breast carcinoma.10Campbell SC Volpert OV Ivanovich M Bouck NP Molecular mediators of angiogenesis in bladder cancer.Cancer Res. 1998; 58: 1298-1304PubMed Google Scholar, 33Bertin N Clezardin P Kubiak R Frappart L Thrombospondin-1 and -2 messenger RNA expression in normal, benign and neoplastic human breast tissues: correlation with prognostic factors, tumor angiogenesis and fibroblastic desmoplasia.Cancer Res. 1997; 57: 396-399PubMed Google Scholar, 34Grossfeld GD Ginsberg DA Stein JP Bochner BH Esrig D Grosher S Dunn M Nichols PW Taylor CR Skinner DG Cote RJ Thrombospondin-1 expression in bladder cancer: association with p53 alterations, tumor angiogenesis and tumor progression.J Natl Cancer Inst. 1997; 89: 219-227Crossref PubMed Scopus (296) Google Scholar, 35Tokunaga T Nakamura M Oshika Y Tsuchida T Kazuno M Fukushima Y Kawai K Abe Y Kijima H Yamazaki H Tamaoki N Ueyama Y Alterations in tumour suppressor gene p53 correlate with inhibition of thrombospondin-1 gene expression in colon cancer cells.Virchows Arch. 1998; 433: 415-418Crossref PubMed Scopus (36) Google Scholar Whereas TSP1 may be down-regulated in tumor cells, relatively high levels of TSP1 and TSP2 mRNA and protein are associated with stromal fibroblasts.33Bertin N Clezardin P Kubiak R Frappart L Thrombospondin-1 and -2 messenger RNA expression in normal, benign and neoplastic human breast tissues: correlation with prognostic factors, tumor angiogenesis and fibroblastic desmoplasia.Cancer Res. 1997; 57: 396-399PubMed Google Scholar, 36Brown LF Guidi AJ Schnitt SJ Van De Water L Iruela-Arispe ML Yeo T-K Tognazzi K Dvorak HF Vascular stroma formation in carcinoma of the breast.Clin Cancer Res. 1999; 5: 1041-1056PubMed Google Scholar In addition, activated monocytes and macrophages contribute TSP1 to the tumor environment as do endothelial cells.37DiPietro LA Polverini PJ Angiogenic macrophages produce the angiogenic inhibitor thrombospondin 1.Am J Pathol. 1993; 143: 678-684PubMed Google Scholar These data raise the possibility that TSP1 produced by stromal cells may function to inhibit tumor growth. If this is the case, then the Thbs1 gene would represent a landscaper for tumor progression.38Kinzler KW Vogelstein B Landscaping the cancer terrain.Science. 1998; 280: 1036-1037Crossref PubMed Scopus (412) Google Scholar Landscaper genes are expressed by cells within the immediate environment of the transformed tumor cells and act to modify their ability to form tumors. Whereas the genetic modifications that occur in tumor cells have been studied extensively, the effect of stromal cell gene expression has been primarily overlooked.39Hanahan D Weinberg RA The hallmarks of cancer.Cell. 2000; 100: 57-70Abstract Full Text Full Text PDF PubMed Scopus (22042) Google Scholar Systemic treatment of tumor-bearing mice with TSP1 or peptides that are derived from the type 1 repeats inhibits experimental tumor growth.28Guo N-H Drutzch HC Inman JK Roberts DD Thrombospondin 1 and type 1 repeat peptides of thrombospondin 1 specifically induce apoptosis of endothelial cells.Cancer Res. 1997; 57: 1735-1742PubMed Google Scholar, 29Guo N-H Krutzsch HC Inman JK Shannon CS Roberts DD Antiproliferative and antitumor activities of D-reversed peptides derived from the second type 1 repeat of thrombospondin-1.J Peptide Res. 1997; 50: 210-221Crossref PubMed Scopus (63) Google Scholar, 40Volpert OV Lawler J Bouck NP A human fibrosarcoma inhibits systemic angiogenesis and the growth of experimental metastases via thrombospondin-1.Proc Natl Acad Sci USA. 1998; 95: 6343-6348Crossref PubMed Scopus (157) Google Scholar Moreover, increased expression of TSP1 in v-Src-transformed NIH 3T3 cells, transformed endothelial cells, human breast adenocarcinoma MDA-MB-435, or human skin carcinoma cells reduces the size and vascular density of the tumors that are produced when these cells are implanted in mice.41Castle VP Dixit VM Polverini PJ Thrombospondin-1 suppresses tumorigenesis and angiogenesis in serum- and anchorage-independent NIH 3T3 cells.Lab Invest. 1997; 77: 51-61PubMed Google Scholar, 42Sheibani N Frazier WA Thrombospondin 1 expression in transformed endothelial cells restores a normal phenotype and suppresses their tumorigenesis.Proc Natl Acad Sci USA. 1995; 92: 6788-6792Crossref PubMed Scopus (149) Google Scholar, 43Weinstat-Saslow DL Zabrenetzky VS VanHoutte K Frazier WA Roberts DD Steeg PS Transfection of thrombospondin 1 complementary DNA into a human breast carcinoma cell line reduces primary tumor growth, metastatic potential and angiogenesis.Cancer Res. 1994; 54: 6504-6511PubMed Google Scholar, 44Bleuel K Popp S Fusenig NE Stanbridge EJ Boukamp P Tumor suppression in human skin carcinoma cells by chromosome 15 transfer or thrombospondin-1 overexpression through halted tumor vasculature.Proc Natl Acad Sci USA. 1999; 96: 2065-2070Crossref PubMed Scopus (106) Google Scholar, 45Streit M Velasco P Brown LF Skobe M Richard L Riccardi L Lawler J Detmar M Overexpression of thrombospondin-1 decreases angiogenesis and inhibits the growth of human cutaneous squamous cell carcinomas.Am J Pathol. 1999; 155: 441-452Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar All of the tumor studies that have been performed to date have investigated the effects of TSP1 on tumors arising from the transplantation of fully transformed cells. To establish a role for TSP1 in the progression of spontaneously occurring tumors arising at orthotopic sites in vivo, we have crossed mice that are deficient in TSP1 with mice that are deficient in p53. Mice that lack TSP1 and are either homozygous or heterozygous for thep53-null allele have a decrease in survival when compared with mice that express wild-type levels of TSP1. Moreover, a decreased incidence of osteosarcoma is observed in p53-deficient mice in the absence of TSP1. We also show that B16F10 and F9 experimental tumors grow more rapidly in Thbs1-null mice. Increased B16F10 tumor cell proliferation and vascular density are observed in the absence of TSP1 whereas the apoptotic index is decreased. These data establish the TSP1 gene as a modifier or landscaper gene for tumor progression. TSP1-deficient mice were crossed with p53-deficient mice that were kindly provided by Dr. Tyler Jacks (Massachusetts Institute of Technology).3Jacks T Remington L Williams BO Schmitt EM Halachmi S Bronson RT Weinberg RA Tumor spectrum analysis in p53-mutant mice.Curr Biol. 1994; 4: 1-7Abstract Full Text Full Text PDF PubMed Scopus (1732) Google Scholar, 46Lawler J Sunday M Thibert V Duquette M George EL Rayburn H Hynes RO Thrombospondin-1 is required for normal murine pulmonary homeostasis and its absence causes pneumonia.J Clin Invest. 1998; 101: 982-992Crossref PubMed Scopus (382) Google Scholar All of the mice in the experiment were derived from two p53-null males and four TSP1-null females. The two p53-null males were littermates from a heterozygous cross. The four TSP1-null females were offspring of a single TSP1-null cross. Both strains of mice were on a mixed C57BL/6J and 129Sv background. Double-heterozygous offspring were used to produce mice that were deficient in both gene products. Double-homozygous males were crossed to females that were homozygous for the Thbs1-null allele and heterozygous for thep53-null allele. The progeny of these crosses were homozygous for the Thbs1-null allele and homozygous or heterozygous for the p53-null allele. Both populations were followed for survival and tumor spectrum. Control groups that were homozygous for the wild-type Thbs1 allele and homozygous or heterozygous for the p53-null allele were established in the same way. All genotyping was done using the polymerase chain reaction (PCR) with DNA prepared from portions of the tail as described by Laird and co-workers.47Laird PW Zijderveld A Linders K Rudnicki MA Jaenisch R Berns A Simplified mammalian DNA isolation procedure.Nucl Acids Res. 1991; 19: 4293Crossref PubMed Scopus (1297) Google Scholar Genotyping for Thbs1 andp53 was done by PCR as described previously.3Jacks T Remington L Williams BO Schmitt EM Halachmi S Bronson RT Weinberg RA Tumor spectrum analysis in p53-mutant mice.Curr Biol. 1994; 4: 1-7Abstract Full Text Full Text PDF PubMed Scopus (1732) Google Scholar, 46Lawler J Sunday M Thibert V Duquette M George EL Rayburn H Hynes RO Thrombospondin-1 is required for normal murine pulmonary homeostasis and its absence causes pneumonia.J Clin Invest. 1998; 101: 982-992Crossref PubMed Scopus (382) Google Scholar A complete necropsy was performed on mice that were found shortly after death or that were sacrificed because they had a tumor burden that was >10% of their body weight, they were moribund, or they displayed poor body condition. A ventral incision was used to open the abdominal and thoracic cavities and the intestines were extended. The top of the skull was removed to facilitate fixation of the brain. The open carcasses were placed in 10% formalin [3.7% formaldehyde in phosphate-buffered saline (PBS)] for fixation and storage until processing for histology. A portion of each organ was embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Survival data were analyzed using the log-rank test for two-way comparisons (Thbs1+/+, p53−/− versus Thbs1−/−, p53−/−, and Thbs1+/+,p53+/− versus Thbs1−/−,p53+/− and two-sided P values are reported.48Miller Jr, RG Survival Analysis. Wiley and Sons, New York1981Google Scholar The incidence of the various tumor types was compared among the four genotypes using the 4 by 2 Pearson's chi-square analysis.49Dallal GE Statools: statistical utility programs.Am Stat. 1986; 40: 236Crossref Google Scholar For determination of theP value, two-way comparisons were used as above andP values were not adjusted for multiple comparisons. The analysis for LOH of the p53 allele was performed on paraffin-embedded 10-μm sections essentially as described by Bianchi and co-workers.50Bianchi AB Navone NM Conti CJ Detection of loss of heterozygosity in formalin-fixed paraffin-embedded tumor specimens by the polymerase chain reaction.Am J Pathol. 1991; 138: 279-284PubMed Google Scholar A probe for mouse p53 was prepared using PCR with the forward primer 5′-ACA GCG TGG TGG TAC CTT AT-3′ and the reverse primer 5′-TAT ACT CAG AGC CGG CCT-3′.3Jacks T Remington L Williams BO Schmitt EM Halachmi S Bronson RT Weinberg RA Tumor spectrum analysis in p53-mutant mice.Curr Biol. 1994; 4: 1-7Abstract Full Text Full Text PDF PubMed Scopus (1732) Google Scholar The PCR product was subcloned into pBluescript KS (Invitrogen, Carlsbad, CA) and used as a probe for Southern blotting of the p53 PCR products.51Maniatis T Fritsch EF Sambrook J Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor1982: 382-389Google Scholar The B16F10 murine melanoma cell line was obtained from the ATCC (Rockville, MD) and cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 50 μg/ml penicillin, 50 U/ml streptomycin, and 2 mmol/L glutamine. The F9 murine testicular teratocarcinoma cell line was obtained from Dr. Lorraine Gudas (Cornell University Medical College) and cultured in 0.3% gelatin-coated dishes in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 50 mg/ml penicillin, and 50 U/ml streptomycin. Five- to 8-week-old C57BL/6J male control mice (Taconic Farms, Germantown, NY) were acclimated, caged in groups of four or less, and their backs were shaved. The Thbs1-null allele was bred to the C57BL/6J background by eight backcrosses to C57BL/6J mice beginning with the chimeric males. Cultured B16F10 or F9 cells (2.5 × 105) were inoculated subcutaneously on the backs of C57BL/6J or 129Sv mice, respectively. Tumors were measured with a dial-caliper and the volumes were determined using the formula width2Donehower LA Harvey M Slagle BL McArthur MJ Montgomery Jr, CA Butel JS Bradley A Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumors.Nature. 1992; 356: 215-221Crossref PubMed Scopus (4020) Google Scholar × length × 0.52. After 16 days, the mice were sacrificed and the tumors were cut and fixed in neutral-buffered formalin. For immunohistochemistry, paraffin-embedded tissue sections were deparaffinized and rehydrated as described previously.52Corless CL Mendoza A Collins T Lawler J Colocalization of thrombospondin and syndecan during murine development.Dev Dyn. 1992; 193: 346-358Crossref PubMed Scopus (64) Google Scholar Sections were treated with 0.25% trypsin in PBS for 30 minutes at 22°C. Monoclonal anti-proliferating cell nuclear antigen (Santa Cruz Biotechnology, Santa Cruz, CA) and anti-CD31 (PharMingen, La Jolla, CA) antibodies were used with the Vectastain ABC kit (Vector Laboratories, Burlingame, CA) to detect proliferating tumor cells or capillaries, respectively. For terminal dUTP nick-end labeling staining, the sections were treated with 20 μg/ml of proteinase K for 15 minutes at 22°C. The slides were washed twice with PBS and incubated in terminal deoxynucleotide transference (TdT) buffer (Life Technologies, Inc., Grand Island, NY) for 10 minutes at 22°C. The buffer was removed and 300 U/ml of TdT (Life Technologies, Inc.) and 2 nmol/L biotin-16–2′-deoxyuridine-5′-triphosphate (Boehringer Mannheim, Indianapolis, IN) was added for 30 minutes at 37°C. The slides were washed three times and developed with the Vectastain ABC kit (Vector Laboratories) ac
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