Although both males and females were used in this experiment, data for males only are shown, as females were not uniformly in telogen at initiation. CD34KO, and this occurred by week 11 in experiment 2 ( 0.02). By the end of each study, the statistical significance had strengthened ( Rabbit Polyclonal to AML1 (phospho-Ser435) 0.0001 and 0.0004 for experiments 1 and 2, respectively) as tumor multiplicity increased in WT mice, whereas CD34KO remained tumor-free. Open in a separate window Physique 2 Papilloma development in DMBA-initiated, TPA-promoted CD34KO mice. Seven-week-old Polydatin (Piceid) CD34KO and WT mice were first initiated with subtumorigenic doses of DMBA and then promoted for 20 weeks with three applications per week of TPA in the standard two-stage mouse skin carcinogenesis regimen. Papillomas were counted weekly. and mice were initiated with 200 nmol DMBA and then promoted thrice weekly with 4 g TPA for 20 wks. average number of tumors per mouse. incidence of papillomas. and CD34KO and WT mice were initiated with 400 nmol DMBA and then promoted thrice weekly with 5 g TPA for 20 wks. average number of tumors per mouse. incidence of papillomas. mice were initiated with either 200 or 400 nmol DMBA, and keratinocytes were harvested 20 h later for DNA isolation and 32P-postlabeling identification of DMBADE DNA adducts. DNA adducts for WT at 200 and 400 nmol; DNA adducts for CD34KO mice Polydatin (Piceid) at 200 and 400 nmol. (retention time, 0.96 min) and (retention time, 1.05 min), respectively. An additional tumor experiment was conducted with CD34KO using C57BL/6 mice as the WT strain, and in this case, the initiating dose of DMBA was increased to Polydatin (Piceid) 400 nmol, and the promoting dose of TPA was increased from 4 to 5 g per application. The purpose here was to compare the CD34KO mouse to an alternate WT strain and to test the effect of a more stringent initiation-promotion regimen on tumor development, given the predominant C57BL/6 background of the KO mice. Although both males and females were used in this experiment, data for males only are shown, as females were not uniformly in telogen at initiation. As shown in Fig. 2and 0.03). Female CD34KO mice also developed a low incidence of tumors (data not shown), although one female developed a single papilloma at week 8 of promotion and went on to develop four papillomas by end of study. Therefore, increasing the dose of DMBA resulted in papilloma development with a significantly increased latency and reduction in overall tumor numbers, confirming an important role for CD34 in tumor development. The results of the tumor experiments described above suggest a critical role for CD34 in either the initiation or promotion stages, although the ability to form at least some tumors implicates a stronger role in promotion. However, because of the lack of a tumor response at the 200 nmol dose of DMBA and the attenuated response at 400 nmol DMBA, we were interested in assessing the ability of the CD34KO mice to metabolically activate DMBA. In order for a papilloma response to be elicited in DMBA-initiated tumorigenesis, DMBA must be metabolically activated by members of the P450 family into DMBADEs (10). DNA adducts form from these metabolites, particularly to the adenine residue in codon 61 in the c-Ha-proto-oncogene, ultimately forming a permanent mutation in (A to T transversion; ref. 10). In general, this is considered to be the causal event in the two-stage skin tumor model, as 90% of DMBA-induced papillomas carry this mutation (42). CD34KO and WT C57BL/6 mice were dosed with either 200 or 400 nmol DMBA and keratinocytes were harvested 24 h after exposure, and then DNA adduct formation was assessed by 32P-postlabeling analysis (Fig. 2H&E-stained, high-magnification photomicrographs of the interfollicular epidermis in WT (basal cells. Compare the simple cuboidal cells of the CD34KO mouse with the irregular layers of larger elongated basal cells of WT mice. and photomicrographs of H&E-stained skin taken 24 and 48 h after the last dose of either 4 5 g TPA or 4 10 g TPA. Bar, 100 m. In WT mice, previously resting hair follicles enter Polydatin (Piceid) the growing stage in response to TPA treatment. In contrast, hair follicles in CD34KO mice remain in telogen, the resting phase of the hair Polydatin (Piceid) cycle. WT images; CD34KO images. Compare the small inactive hair follicles and thin epidermis of CD34KO mice with the large active growing hair follicles.
Although both males and females were used in this experiment, data for males only are shown, as females were not uniformly in telogen at initiation