Aberrant cell cycle progression contributes to the early-stage accelerated carcinogenesis in transgenic epidermis expressing the dominant negative TGFβRII

Abstract

Mutations in the transforming growth factor b type II receptor (TGFbRII) have been found in various malignant tumors, suggesting that loss of TGFb signaling plays a causal role in late-stage cancer development. To test whether loss of TGFbRII is involved in early-stage carcinogenesis, we have generated transgenic mice expressing a dominant negative TGFbRII (DbRII) in the epidermis. These mice exhibited an increased susceptibility to chemical carcinogenesis protocols at both early and late stages. In the current study, parameters for cell cycle progression and chromosome instability were analysed in DbRII tumors. DbRII papillomas showed an increased S phase in¯ow cytometry. Bromodeoxyuridine (BrdU) labeling and mitotic indices in DbRII papillomas also showed a threefold increase compared to papillomas developing in non-transgenic mice. When papillomas further progressed to squamous cell carcinomas (SCC), both control and DbRII SCC showed similar BrdU labeling indices and percentages of S phase cells. However, DbRII SCC cells showed a sixfold increase in the G2/M population. Mitotic indices in DbRII SCC also showed a threefold increase compared to non-transgenic SCC. Consistent with a perturbed cell cycle, DbRII papillomas and SCC showed reduced expression of the TGFb target genes p15 (INK4b), p21 (WAF-1) and p27 (Kip1), inhibitors of cyclin-dependent kinases (cdks). However, most DbRII papilloma cells exhibited normal centrosome numbers, and DbRII SCC exhibited a similar extent of centrosome abnormalities compared to control SCC (35 ± 40% cells). Most of DbRII SCC exhibited diploid chromosome pro®les. These data indicate that inactivation of TGFbRII accelerates skin tumorigenesis at early stages by the acceleration of loss of cell cycle control, but not by increased chromosome instability. Oncogene (2000) 19, 3623 ± 3631.

Epithelial stem cell mutations that promote squamous cell carcinoma metastasis

“…A tumor cell suspension was prepared as described (64). Briefly, tumors were harvested from mice, chopped into small pieces, and digested in a collagenase solution at 37°C for 30 minutes.…”

“…Tumor cell lines were generated as we previously described (64). Briefly, tumor pieces were digested in collagenase (Worthington) for 45 minutes at 37°C and in 0.25% trypsin for 15 minutes at 37°C.…”

Distinct mechanisms of TGF- 1-mediated epithelial-to-mesenchymal transition and metastasis during skin carcinogenesis

“…into these mice. Papillomas and adjacent skin from TGF-β1-transgenic and control mice were dissected 2 hours after the injection, fixed, and processed as previously described (48). Sections were incubated with an FITC-conjugated monoclonal antibody to BrdU (BD Biosciences) and a guinea-pig antiserum to mouse keratin 14 (K14), which reacts with the epithelial component of papillomas.…”

Smad3 Knockout Mice Exhibit a Resistance to Skin Chemical Carcinogenesis

“…In analyzing the relative expression levels of other genes, the expression level from one Smad3 Ϫ/Ϫ sample (unless otherwise specified) of each particular gene being analyzed was set as 1 arbitrary unit. To examine expression levels of various cell cycle control genes, RNase protection assay was performed using RNase protection assay III kits (Ambion), as we have described previously (31). The probes used included multiprobe gene sets [mCC1c, mCYC-1, and mCYC-2 (BD Biosciences)] and individual probes for c-myc, p15, and p21 (31).…”