We developed a procedure for growing normal epidermal keratinocyte stem cells isolated from a single punch biopsy of adult human skin in long-term culture. Primary skin epithelial cells were maintained in collagen-coated plates with irradiated human neonatal foreskin fibroblasts (line HPI.1) as a feeder for more than 120 days, approximately 115 population doublings, without signs of replicative senescence. Clonal analysis revealed the presence of holoclones, meroclones, and paraclones. Only emerging colonies with high proliferative potentials and extensive capacities for division (holoclones and meroclones) were subcultured, favoring the expansion of stem cells and progenitors capable of prolonged self-maintenance when subcloned, thus accounting for the prevailing long-term proliferation of the original culture. We found that meroclones included bipotent progenitors capable of generating both keratinocytes and mucin-producing cells. The numbers of these cells were greater after confluence, suggesting that commitment for their differentiation occurred late in the life of a single clone. On a three-dimensional gelatin matrix and on a collagen layer containing the fibroblast feeder, cells isolated from the expansion of holoclones and meroclones formed stratified cohesive layers of keratinocytes that were able to further differentiate, as in normal skin. These results indicate that our procedure will serve as a valuable tool to study expansion of epidermal stem cells as well as the growth mechanisms and cell products associated with their growth and differentiation.

No abstract

Objective: The objective of the study was to evaluate the expression and functional activity of Peroxisome proliferator-activated receptor (PPAR) g in pituitary adenomas from 14 consecutive acromegalic patients and to establish its role in apoptosis. Subjects and methods: Fourteen consecutive acromegalic patients were enrolled in the study. Wistar-Furth rats were used for in vivo studies. Expression of PPARg was evaluated by RT-PCR and Western blot. Apoptosis and cell cycle were assessed by FACS analysis. The effects of PPARg ligands on transcriptional regulation of GH gene were evaluated by RT-PCR and electromobility shift assay. Results: PPARg was expressed in all human GH-secreting adenoma (GH-oma), in normal pituitary tissue samples (39^24% and 78^5% of immunostained nuclei respectively; P , 0.0002; ANOVA), and in rat GH-secreting (GH3) cells. A PPRE-containing reporter plasmid transfected into GH3 cells was activated by ciglitazone or rosiglitazone (TZDs), indicating that PPARg was functionally active. Treatment of GH3 cells with TZDs increased apoptosis in a dose-dependent manner (P ¼ 0.0003) and arrested cell proliferation, reducing the number of cells in the S-phase (P , 0.0001 vs untreated cells). TZDs increased the expression of TRAIL, leaving unaffected that of p53 and Bax. TZDs reduced GH concentrations in the culture media from 43.7^5.4 ng/ml to 2.1^0.3 ng/ml (P , 0.0001) and in cell extracts (P , 0.004). PPARg-RXRa heterodimers bound to GH promoter, inhibiting its activity and reducing GH mRNA levels (1.8 £ 10 6 vs 5.7 £ 10 6 transcripts respectively vs untreated cells; P , 0.002). Subcutaneous GH-oma developed in rats injected with GH3 cells; tumor growth increased in placebo-treated rats and to a lesser extent in TZDs-treated animals (24.1^2.0 g, and 14.8^4.2 g respectively, P , 0.03). Serum GH concentrations were lower in TZDs-treated rats than in controls (871^67 ng/ml vs 1.309^238 ng/ml; P , 0.05). Conclusions:The results of this study indicate that PPARg controls GH transcription and secretion as well as apoptosis and growth of GH-oma; thus, TZDs have the potential of a useful tool in the complex therapeutic management of acromegalic patients.

Multifunctional cytokines play important and only partially defined roles in mammary tumour development and progression. Normal human mammary epithelial cells constitutively produce interleukin 6 (IL-6), IL-8 and a non-secreted form of tumour necrosis factor. Transformation of mammary epithelial cells by different oncogenes is frequently associated with alterations of cytokine/growth factor production and responsiveness. In the present study we analysed the expression of IL-6 in 149 cases of invasive breast carcinoma and the data have been correlated with clinico-pathological variables including tumour size, histological grade, nodal status, and oestrogen and progesterone receptors, Ki67 and p53, protein expression. Though the majority of breast carcinomas expressed at least low levels of immunoreactive IL-6, we found that expression of this cytokine was inversely associated with histological tumour grade ( P = 0.0017), but not with tumour size and nodal status. Ki67 positivity was inversely correlated with IL-6 expression ( P = 0.027). Among biological parameters analysed, a direct association was found between the percentage of IL-6-positive cells and that of oestrogen ( P = 0.00005) and progesterone ( P = 0.025) receptor-positive cells. No correlation was observed between IL-6 and p53 protein expression. These data indicate that down-regulation of IL-6 is associated with highly malignant mammary carcinomas. It will be of interest to evaluate whether alterations of cytokines that are constitutively produced by mammary cells are also associated with high-grade tumours. © 1999 Cancer Research Campaign

Apoptosis is important for both tissue development and differentiation; its deregulation may contribute to tumourigenesis. In order to clarify the role of Bcl-2, an apoptosis-inhibiting protein, in pancreatic morphogenesis and tumour progression, its immunohistochemical expression was evaluated in 12 samples of fetal pancreas, in 10 samples of adult pancreas with ductal hyperplastic lesions, in 120 cases of primary pancreatic ductal adenocarcinoma, and in 43 synchronous metastatic lymph nodes. To evaluate the role of apoptosis in pancreatic cancer, p53 expression was also studied in tumour samples. Bcl-2 cytoplasmic acinar and ductal immunostaining was found in all fetal and adult tissue samples; ductal hyperplastic lesions were constantly negative. Thirty out of 120 (25%) tumours and 3 out of 43 (7%) lymph nodes expressed Bcl-2, whereas 67 out of 120 (56%) expressed nuclear p53. Well-differentiated tumours (G1) were more frequently Bcl-2-positive (p=0.002); furthermore, there was an inverse correlation between Bcl-2 and p53 expression in primary tumours (p=0.02). Neither Bcl-2 nor p53 influenced patients' prognosis, which was instead affected by N (p=0.02) and M (p<0.0001) status and stage of the disease (p=0.002). It is concluded that Bcl-2 regulates pancreatic morphogenesis and tissue homeostasis from early fetal to adult life and can be considered a phenotypic marker of normal exocrine pancreas. On the other hand, the lack of expression in preneoplastic lesions and the low positivity found in primary tumours and lymph node metastases suggest that Bcl-2 does not play a centralrole in pancreatic tumourigenesis and cancer progression.