Keratinocyte Expression of MMP3 Enhances Differentiation and Prevents Tumor Establishment
2008; Elsevier BV; Volume: 173; Issue: 5 Linguagem: Inglês
10.2353/ajpath.2008.080132
ISSN1525-2191
AutoresLisa J. McCawley, Jane Wright, Bonnie LaFleur, Howard C. Crawford, Lynn M. Matrisian,
Tópico(s)Bone and Dental Protein Studies
ResumoMatrix metalloproteinase (MMP)−3 is induced by multiple cell types in the skin during processes involved in both normal and pathological tissue remodeling. We previously demonstrated that MMP3-null animals have an increased sensitivity to the development of squamous cell carcinoma, suggesting that overall, MMP3 has a protective role in squamous cell carcinoma. However, not all cellular responses affected by a loss of MMP3 are tumor-protective, and tumor expression of MMP3 is co-incident with an invasive tumor phenotype. Transgenic mice were generated with MMP3 targeted to keratinocytes to examine the biological role of tumor-produced MMP3. Overexpression of MMP3 reduced tumor multiplicity in response to chemically induced squamous cell carcinoma. Vascular density was increased with MMP3 overexpression; however, other cellular processes, including tumor growth and leukocyte infiltration, were unaffected. In accordance with the change in tumor multiplicity, SP-1 murine papilloma cell lines that were generated to stably express MMP3 lost the capacity to establish palpable tumors following orthotopic injection into immunocompromised mice. Analysis of epidermal biopsies taken at 1 to 2 weeks postinjection revealed that these MMP3-expressing Sp-1 lines had reduced levels of proliferation and pronounced differentiation. These same cells demonstrated an increased ability to differentiate in vitro, an effect that was inhibited by broad-spectrum MMP and selective MMP3 inhibition. These studies suggest that keratinocyte expression of MMP3 promotes cellular differentiation, impeding tumor establishment during tumorigenesis. Matrix metalloproteinase (MMP)−3 is induced by multiple cell types in the skin during processes involved in both normal and pathological tissue remodeling. We previously demonstrated that MMP3-null animals have an increased sensitivity to the development of squamous cell carcinoma, suggesting that overall, MMP3 has a protective role in squamous cell carcinoma. However, not all cellular responses affected by a loss of MMP3 are tumor-protective, and tumor expression of MMP3 is co-incident with an invasive tumor phenotype. Transgenic mice were generated with MMP3 targeted to keratinocytes to examine the biological role of tumor-produced MMP3. Overexpression of MMP3 reduced tumor multiplicity in response to chemically induced squamous cell carcinoma. Vascular density was increased with MMP3 overexpression; however, other cellular processes, including tumor growth and leukocyte infiltration, were unaffected. In accordance with the change in tumor multiplicity, SP-1 murine papilloma cell lines that were generated to stably express MMP3 lost the capacity to establish palpable tumors following orthotopic injection into immunocompromised mice. Analysis of epidermal biopsies taken at 1 to 2 weeks postinjection revealed that these MMP3-expressing Sp-1 lines had reduced levels of proliferation and pronounced differentiation. These same cells demonstrated an increased ability to differentiate in vitro, an effect that was inhibited by broad-spectrum MMP and selective MMP3 inhibition. These studies suggest that keratinocyte expression of MMP3 promotes cellular differentiation, impeding tumor establishment during tumorigenesis. Matrix metalloproteinases (MMPs) are a family of extracellular matrix-degrading proteinases implicated in a variety of normal and pathological cellular processes including embryogenesis, angiogenesis, wound healing and cancer.1Parks WC Wilson CL Lopez-Boado YS Matrix metalloproteinases as modulators of inflammation and innate immunity.Nat Rev Immunol. 2004; 4: 617-629Crossref PubMed Scopus (1426) Google Scholar, 2Brinckerhoff CE Matrisian LM Matrix metalloproteinases: a tail of a frog that became a prince.Nat Rev Mol Cell Biol. 2002; 3: 207-214Crossref PubMed Scopus (953) Google Scholar While key roles for these enzymes in regulating fundamental processes throughout tumor progression and tumor invasion have been delineated3McCawley LJ Matrisian LM Matrix metalloproteinases: they're not just for matrix anymore!.Curr Opin Cell Biol. 2001; 13: 534-540Crossref PubMed Scopus (1081) Google Scholar, 4Egeblad M Werb Z New functions for the matrix metalloproteinases in cancer progression.Nat Rev Cancer. 2002; 2: 161-174Crossref PubMed Scopus (5031) Google Scholar, 5Page-McCaw A Ewald AJ Werb Z Matrix metalloproteinases and the regulation of tissue remodelling.Nat Rev Mol Cell Biol. 2007; 8: 221-233Crossref PubMed Scopus (2121) Google Scholar; there has been a lack of efficacy with broad-spectrum inhibition of MMPs as anti-tumor therapy.6Coussens LM Fingleton B Matrisian LM Matrix metalloproteinase inhibitors and cancer: trials and tribulations.Science. 2002; 295: 2387-2392Crossref PubMed Scopus (2331) Google Scholar During tumorigenesis, the expression of a number of MMPs are induced de novo.7Hanahan D Weinberg RA The hallmarks of cancer.Cell. 2000; 100: 57-70Abstract Full Text Full Text PDF PubMed Scopus (21638) Google Scholar Recent work supports the idea that MMPs, themselves, may play beneficial, anti-tumor roles throughout tumorigenesis.8Martin MD Matrisian LM The other side of MMPs: protective roles in tumor progression.Cancer Metastasis Rev. 2007; 26: 717-724Crossref PubMed Scopus (246) Google Scholar, 9Lopez-Otin C Matrisian LM Emerging roles of proteases in tumour suppression.Nat Rev Cancer. 2007; 7: 800-808Crossref PubMed Scopus (634) Google Scholar As these same MMPs can regulate cellular processes required during normal tissue remodeling, it is difficult to distinguish by association between MMPs that have been selected for pro-tumorigenic impulse and those that reflect a counter response attempting to initiate tissue repair and maintain homeostasis.10Radisky D Hagios C Bissell MJ Tumors are unique organs defined by abnormal signaling and context.Semin Cancer Biol. 2001; 11: 87-95Crossref PubMed Scopus (150) Google Scholar Thus, a renewed focus has begun on defining the role of individual proteinases throughout tumor progression to determine the distinct spatial and temporal functions of these enzymes and to better predict efficacious modulation of these proteinases for anti-tumor therapy.11Overall CM Kleifeld O Tumour microenvironment - opinion: validating matrix metalloproteinases as drug targets and anti-targets for cancer therapy.Nat Rev Cancer. 2006; 6: 227-239Crossref PubMed Scopus (1004) Google Scholar, 12Hu J Van den Steen PE Sang QX Opdenakker G Matrix metalloproteinase inhibitors as therapy for inflammatory and vascular diseases.Nat Rev Drug Discov. 2007; 6: 480-498Crossref PubMed Scopus (644) Google Scholar There is correlative evidence suggesting that epidermal expression of one MMP family member, MMP3 (Stromelysin-1/E.C.3.4.24.17), influences tumor invasion. Under resting conditions, the skin produces negligible amounts of MMP3. However, MMP3 is transiently up-regulated in both the dermis and epidermis during normal re-epithelialization and, pathologically, in chronic wounds, blistering skin diseases and squamous cell carcinoma (SCC).13Madlener M Parks WC Werner S Matrix metalloproteinases (MMPs) and their physiological inhibitors (TIMPs) are differentially expressed during excisional skin wound repair.Exp Cell Res. 1998; 242: 201-210Crossref PubMed Scopus (281) Google Scholar, 14Saarialho-Kere UK Vaalamo M Airola K Niemi KM Oikarinen AI Parks WC Interstitial collagenase is expressed by keratinocytes that are actively involved in reepithelialization in blistering skin diseases.J Invest Dermatol. 1995; 104: 982-988Crossref PubMed Scopus (66) Google Scholar, 15Saarialho-Kere UK Pentland AP Birkedal-Hansen H Parks WC Welgus HG Distinct populations of basal keratinocytes express stromelysin-1 and stromelysin-2 in chronic wounds.J Clin Invest. 1994; 94: 79-88Crossref PubMed Scopus (203) Google Scholar, 16Weckroth M Vaheri A Lauharanta J Sorsa T Konttinen YT Matrix metalloproteinases, gelatinase and collagenase, in chronic leg ulcers.J Invest Dermatol. 1996; 106: 1119-1124Crossref PubMed Scopus (215) Google Scholar, 17Kerkela E Saarialho-Kere U Matrix metalloproteinases in tumor progression: focus on basal and squamous cell skin cancer.Exp Dermatol. 2003; 12: 109-125Crossref PubMed Scopus (267) Google Scholar Whereas MMP3 expression is induced in the tumor stroma in the early stages of tumorigenesis, tumor expression of MMP3 is co-incident with the development of highly invasive SCC and progression to an epithelial-to-mesenchymal phenotype.17Kerkela E Saarialho-Kere U Matrix metalloproteinases in tumor progression: focus on basal and squamous cell skin cancer.Exp Dermatol. 2003; 12: 109-125Crossref PubMed Scopus (267) Google Scholar, 18Nagase H Stromelysins 1 and 2.in: Parks WC Mecham RP Matrix metalloproteinases. 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Despite the evidence for an MMP3-dependent pro-tumorigenic role, our previous study indicated that the complete absence of MMP3 results in mice with an enhanced sensitivity to chemically induced SCC suggesting that MMP3 is overall anti-tumorigenic during SCC progression.27McCawley LJ Crawford HC King Jr, LE Mudgett J Matrisian LM A protective role for matrix metalloproteinase-3 in squamous cell carcinoma.Cancer Res. 2004; 64: 6965-6972Crossref PubMed Scopus (118) Google Scholar However, not all cellular responses affected by loss of MMP3 are associated with a tumor protective role. Furthermore, as MMP3 is expressed by a variety of cell types and primarily by cells residing in the tumor stroma at early stages of SCC, the relative contribution of MMP3 from discrete cell populations is not readily discernable.28Abramson SR Conner GE Nagase H Neuhaus I Woessner Jr, JF Characterization of rat uterine matrilysin and its cDNA Relationship to human pump-1 and activation of procollagenases.J Biol Chem. 1995; 270: 16016-16022Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar Due to the variety of potential targets, we anticipate that there are both distinct protective and pro-tumorigenic MMP3-dependent responses that will depend on the source and localization of MMP3 production. In view of the fact that tumor expression of MMP3 is co-incident with gain of epithelial-to-mesenchymal phenotype, the association of MMP3 expression with highly invasive tumorigenic keratinocytes and the potential for pro-invasive effects following proteolytic digestion of select targets, we predicted that tumor localized expression of MMP3 would be tumor promoting. To begin to understand the role of keratinocyte-expression of MMP3, we took an approach complementary to the MMP3 null study and generated a transgenic mouse with epidermal targeted expression of MMP3 and SCC cell lines with stable MMP3 introduction. We have compared, in vivo, the tumorigenic responses of wild-type (WT) and MMP3 transgenic animals and the tumorigenic properties of squamous cell lines with MMP3 overexpression. Transgenic mice with targeted expression of MMP3 were generated using a vector containing the bovine cytokeratin 5 and 6 minilocus obtained from Manfred Blessing.29Blessing M Nanney LB King LE Jones CM Hogan BL Transgenic mice as a model to study the role of TGF-beta-related molecules in hair follicles.Genes Dev. 1993; 7: 204-215Crossref PubMed Scopus (154) Google Scholar This vector contains endogenous splice and Poly A addition sites. A construct, rMMP3301 containing the full length WT rat MMP3 cDNA with an amino acid substitution at position 93 (P→V) was inserted into a SalI site in the vector placing the cDNAs under the control of the K5 promoter. This amino acid substitution results in autoactivated form of MMP3.30Park AJ Matrisian LM Kells AF Pearson R Yuan Z Navre M Mutational analysis of the transin (rat stromelysin) autoinhibitor region demonstrates a role for residues surrounding the "cysteine switch.".J Biol Chem. 1991; 266: 1584-1590Abstract Full Text PDF PubMed Google Scholar The vector sequences were isolated with a BamHI digestion, purified by CsCl centrifugation and microinjected into (C57Bl/6xDBA) F1 fertilized eggs as previously described.31Witty JP Wright J Matrisian LM Matrix metalloproteinases are expressed during ductal and alveolar mammary morphogenesis, and misregulation of stromelysin-1 in transgenic mice induces unscheduled alveolar development.Mol Biol Cell. 1995; 6: 1287-1303Crossref PubMed Scopus (169) Google Scholar Embryos were transferred to pseudopregnant females and pups were analyzed for presence of the transgene by Southern blotting. Transgenic founder animals were identified by Southern blot analysis of EcoRI-digested tail DNA under high stringency conditions as previously described.31Witty JP Wright J Matrisian LM Matrix metalloproteinases are expressed during ductal and alveolar mammary morphogenesis, and misregulation of stromelysin-1 in transgenic mice induces unscheduled alveolar development.Mol Biol Cell. 1995; 6: 1287-1303Crossref PubMed Scopus (169) Google Scholar The number of copies of transgene DNA that integrated into the genome was determined by comparing the relative intensity of the hybridization signal to control DNAs containing 1 and 10 genome equivalents of the same DNA that was injected. Transgenic lines were generated by mating founder animals to (C57Bl/6xDBA) F1 males and females. Tail DNA samples were harvested for genotyping using PCR. We used a sense oligonucleotide primer (5′-TGAAGGTCTGGGAGGAGGTGAC-3′) and an antisense primer (5′-TTCCAGGCCCATCAAAAGGGA-3′). The resulting amplified fragment spans exon 4 and exon 5 and results in a ∼260-bp fragment from the genomic DNA and a ∼160-bp fragment from the rMMP3 transgene. Transgenic and litter matched control mice were anesthetized with ketamine/xylazine and had their dorsal fur shaved. Full thickness excisional wounds were made using 3-mm biopsy punches (Acuderm, Ft. Lauderdale Florida). Skin samples were harvested and fixed in 4% paraformaldehyde and paraffin embedded. Chemically induced SCC experiments were performed on C57Bl/6xDBA heterozygous transgenic and litter matched controls as previously described.27McCawley LJ Crawford HC King Jr, LE Mudgett J Matrisian LM A protective role for matrix metalloproteinase-3 in squamous cell carcinoma.Cancer Res. 2004; 64: 6965-6972Crossref PubMed Scopus (118) Google Scholar Mice were housed, fed and treated in accordance with the guidelines approved by the Committee for Protection of Animal Subjects at Vanderbilt University Medical Center. At 8 weeks of age, K5*-MMP3301.24 transgenic and WT litter mate control mice were shaved on the dorsal area 2 days before initiating chemical carcinogenesis treatments and thereafter as needed. Mice were subjected to a single topical application of a solution containing 25 μg 7,12-dimethylbenz [a] anthracene (DMBA; Sigma Chemical Co., St. Louis, MO) dissolved in 100 μl of acetone directly applied to shaved skin. One week after the first treatment, 5 μg 12-O-tetradecanoylphorbol-13-acetate; (TPA; LC Laboratories, Woburns, MA) dissolved in 100 μl of acetone was applied twice weekly for 25 weeks and terminated 48 hours before tumors were harvested. Mice were examined weekly for the presence of skin tumors and for tumor dimensions to be recorded. Tumor volumes were estimated according to the formula V = (L/2) × (W)2Brinckerhoff CE Matrisian LM Matrix metalloproteinases: a tail of a frog that became a prince.Nat Rev Mol Cell Biol. 2002; 3: 207-214Crossref PubMed Scopus (953) Google Scholar where V = volume, L = length and W = width. At autopsy, lungs were inflated and fixed with Bouin's fixative and visually examined for the presence of metastases. Squamous tumors were dissected and immediately fixed in 4% paraformaldehyde before paraffin embedding.32Wright JH McDonnell S Portella G Bowden GT Balmain A Matrisian LM A switch from stromal to tumor cell expression of stromelysin-1 mRNA associated with the conversion of squamous to spindle carcinomas during mouse skin tumor progression.Mol Carcinog. 1994; 10: 207-215Crossref PubMed Scopus (58) Google Scholar For orthotopic injections, 8-week-old Rag-2 null mice in the 129SvEv background (Taconic) were subjected to 3 Gy of irradiation by Cesium125 exposure as previously detailed to improve tumor take.33Strickland JE Greenhalgh DA Koceva-Chylan A Hennings H Resterpo C Balaschak M Yuspa SH Development of murine epidermal cell lines which contain an activated ras Ha oncogene and form papillomas in skin grafts on athymic nude mouse hosts.Cancer Res. 1988; 48: 165-169PubMed Google Scholar Sp-1 cells were stably selected following transfection with an expression vector containing the neomycin resistance gene or a vector containing the rMMP3 cDNA as previously detailed.34McDonnell S Matrisian LM Stromelysin in tumor progression and invasion.Cancer Metastasis Rev. 1991; 9: 305-319Crossref Scopus (105) Google Scholar Sp-1 parental cells, empty vector control clones (Sp-1 Neoa1, Neoa2, or Neoa3) or MMP3 expressing clones(Sp-1MMP3a4, MMP3a6 or MMP3a8), 3 × 105 cells diluted in 50 μl of 1× PBS, were introduced by intradermal injection. For the initial tumorigenecity study, mice were examined weekly for presence of skin tumors up to 3 months postinjections. For short term experiments (1 to 2 weeks), mice were tattooed around the site of injection and tissue resected within the tattooed area. For 5-bromo-2′-deoxyuridine (BrdU) incorporation analysis, mice were injected with 75 mg/kg BrdU 2 hours before animals were sacrificed. Tumors were dissected and immediately fixed in 4% paraformaldehyde before paraffin embedding.32Wright JH McDonnell S Portella G Bowden GT Balmain A Matrisian LM A switch from stromal to tumor cell expression of stromelysin-1 mRNA associated with the conversion of squamous to spindle carcinomas during mouse skin tumor progression.Mol Carcinog. 1994; 10: 207-215Crossref PubMed Scopus (58) Google Scholar Paraformaldehyde fixed-paraffin embedded sections (5 μm) of normal, wounded and tumorigenic skin were analyzed by in situ hybridization for rat MMP3 transgene expression as previously described.31Witty JP Wright J Matrisian LM Matrix metalloproteinases are expressed during ductal and alveolar mammary morphogenesis, and misregulation of stromelysin-1 in transgenic mice induces unscheduled alveolar development.Mol Biol Cell. 1995; 6: 1287-1303Crossref PubMed Scopus (169) Google Scholar A 492-bp BglII/HincII fragment corresponding to sequences in the 3′ region of the rat cDNA was used as template in the generation of S35-labeled sense and antisense riboprobes. Paraformaldehyde-fixed paraffin embedded sections (5 μm) were stained with Mayer's H&E or with Trichrome by Gomori's method or analyzed by immunohistochemistry as previously described.27McCawley LJ Crawford HC King Jr, LE Mudgett J Matrisian LM A protective role for matrix metalloproteinase-3 in squamous cell carcinoma.Cancer Res. 2004; 64: 6965-6972Crossref PubMed Scopus (118) Google Scholar The following primary antibodies were used in conjunction with antigen retrieval by heat denaturation (10 minutes microwave, 10 mmol/L sodium citrate, pH 6.0): rabbit polyclonal anti-keratin 5 (1:1000; Covance Research Products Inc., Denver, PA); rabbit polyclonal anti-keratin 6 (1:1000; Covance Research Products Inc., Denver, PA); mouse ascites anti-pan keratin (1:400; Sigma-Aldrich Corp., St. Louis, MO); rabbit polyclonal anti-mouse involucrin (1:400; Covance Research Products Inc., Denver, PA); rabbit polyclonal anti-fillagrin (1:400; Covance Research Products Inc., Denver, PA); mouse monoclonal anti-proliferating cell nuclear antigen (PCNA, 1:400; Zymed Laboratories, San Francisco, CA); rat monoclonal anti-neutrophil (1:200; Serotec Inc., Raleigh, NC); rat monoclonal anti-CD3ε (1:200; Serotec Inc., Raleigh, NC); and mouse anti-smooth muscle actin (1:800; Sigma-Aldrich Corp., St. Louis, MO). The following primary antibodies were used in conjunction with antigen retrieval by proteolysis (10 minutes, 0.1% Trypsin in 10 mmol/L Tris-Cl, pH 7.4) rat monoclonal anti-F4/80 (1:200; Serotec Inc., Raleigh, NC); and rat monoclonal anti-CD31/Pecam (1:100; B.D. Biosciences/Pharmingen. San Jose, CA). For BrdU analysis, the rat anti-BrdU (1:800; Accurate Chemical and Scientific Corp) was used following acid denaturation and neutralization as the antigen retrieval method. Negative controls were performed using appropriate species and isotype matched immunoglobulins. Sections were then incubated with appropriate secondary antibody (Vector Laboratories, Burlingame, CA). For immunohistochemistry, antibody binding was detected using ABC Elite Method (Vector Laboratories, Burlingame, CA) with diaminobenzidine as the substrate and the sections were counter stained with Mayer's Hematoxylin to visualize cells. For immunofluorescence, sections were then incubated with appropriate secondary antibody (Vector Laboratories, Burlingame, CA) and with Hoechst 30551 to visualize cell nuclei. Sections were evaluated for presence or absence of antigen. In the case of PCNA immunostaining, an average of 1000 nuclei were evaluated for 6 to 8 stage matched tumors/experimental group. Quantitation of PECAM/CD-31 immunostaining was performed morphometrically using Metamorph Imaging System software (MDS, Inc, Toronto, Canada) to determine the area of positive staining for 6 to 8 stage matched tumors/experimental group. Neutrophil immunostaining was assessed by counting neutrophil positive cells within multiple arbitrary areas defined by Metamorph Imaging System Software (Universal Imaging Corporation, Downingtown, PA) of 6 to 8 tumors/experimental group. Terminal dUTP nicked-end labeling (TUNEL) was performed on paraformaldehyde-fixed, paraffin embedded sections (5 μm) using the Apo-Tag Kit (Intergen, Purchase, NY) according to manufacturer's directions as previously described.27McCawley LJ Crawford HC King Jr, LE Mudgett J Matrisian LM A protective role for matrix metalloproteinase-3 in squamous cell carcinoma.Cancer Res. 2004; 64: 6965-6972Crossref PubMed Scopus (118) Google Scholar Antibody binding was detected using ABC Elite Method (Vector Laboratories, Burlingame, CA) with diaminobenzidine as the substrate, and sections were counter stained with contrast green to visualize cells. An average of 1500 nuclei were evaluated for TUNEL positivity for 6 to 8 stage-matched tumors/experimental group. For growth of cells in a semisolid media to induce differentiation, cells were resuspended 1 × 105 cells/ml in complete media (10% fetal bovine serum, Dulbecco's modified Eagle's medium), containing 1.65% methylcellulose (Sigma-Aldrich Corp., St. Louis, MO).35Li ER Owens DM Djian P Watt FM Expression of involucrin in normal, hyperproliferative and neoplastic mouse keratinocytes.Exp Dermatol. 2000; 9: 431-438Crossref PubMed Scopus (52) Google Scholar Cells were plated onto polyHEMA coated bacterial dishes (Sigma-Aldrich Corp., St. Louis, MO) and cultured for 24 to 48 hours as indicated in the presence or absence of MMP-3 inhibitor (10 μm N-isobutyl-N-(4-methoxyphenylsulfonyl)-glycylhyrdroxamic acid; Calbiochem). Cells were harvested by washing 3 times in 1× PBS. An aliquot of cells were recovered both before and following growth in suspension. These cells were attached to coverglass by cytospin, and fixed in 10% phosphate buffered formalin for 10 minutes, at room temperature. Cells were immunostained for involucrin to identify differentiation status and with Hoechst 30551 to visualize cell nuclei as described above. Kaplan-Maier plots of time to onset of first tumor were analyzed using the log-rank test. Tumor multiplicity was analyzed by multivariant regression of the type described by Prentice et al.36Prentice RL Williams BJ Peterson AV On the Regression-Analysis of Multivariate Failure Time Data.Biometrika. 1981; 68: 373-379Crossref Scopus (876) Google Scholar Tumor growth was plotted as the change in volume weekly. The linear range was defined as the rate between first appearance of tumors and the week at which the first plateau of each curve was reached. Immunohistochemical results were analyzed by a non-parametrical (Mann-Whitney test) where indicated. All statistical analysis was performed using Statview software (SAS Institute). To assess the role of tumor localized expression of MMP3 during SCC in an autochthanous tumor model, we generated transgenic mice with keratinocyte-targeted MMP3 expression. To target expression of MMP3 to the mouse epidermis, we made a construct by inserting the rat MMP3301 cDNA into the bovine keratin (BK) 5 expression vector as shown in Figure 1A. This particular construct contains an amino acid substitution that results in autoactivated form of MMP3, which bypasses any requirement for endogenous factors for activation of MMP3 to its mature form30Park AJ Matrisian LM Kells AF Pearson R Yuan Z Navre M Mutational analysis of the transin (rat stromelysin) autoinhibitor region demonstrates a role for residues surrounding the "cysteine switch.".J Biol Chem. 1991; 266: 1584-1590Abstract Full Text PDF PubMed Google Scholar; furthermore, we previously used this MMP3 construct to successfully express MMP3 in mammary epithelium.37Witty JP Lempka T Coffey Jr, RJ Matrisian
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