PE
Excitation: 565nm, Emission: 578nm
We have previously introduced the concept of high proliferative potential-quiescent (HPP-Q) cells to refer to primitive human hematopoietic progenitors, on which transforming growth factor-beta1 (TGF-beta1) exerts a pleiotropic effect. TGF-beta1 confers to these slow-dividing cells a mitogenic receptor(low) phenotype and maintains immature properties by preventing differentiation and apoptosis. However, the effect of TGF-beta1 on long-term expansion has not yet been clearly demonstrated. Here, we describe the characterization of a human skin keratinocyte subpopulation, highly enriched for primitive epidermal precursors, on the basis of high adhesion capacity (Adh+++) and low expression of the epidermal growth factor receptor (Adh+++EGF-Rlow). In our standard culture condition without feeder cells, the mean estimated output for cells from an unfractionated population of primary foreskin keratinocytes was 10(7)-10(8), increasing to 10(12)-10(13) in cultures initiated with selected Adh+++EGF-Rlow precursors. Characterization of these cells revealed a hitherto unknown property of TGF-beta1: its addition at a very low concentration (10 pg/ml) in long-term cultures induces a very significant additional increase of expansion. In this optimized system, outputs obtained in cultures initiated with Adh+++EGF-Rlow cells repeatedly reached 10(16)-10(17) ( approximately 60 population doublings, approximately 4 x 10(18) keratinocytes produced per clonogenic cell present in the initial population). At the molecular level, this effect is associated with an increase in Smad1, Smad2 and Smad3 phosphorylation and an increase in alpha6 and beta1 integrin expression. No such effect could be observed on mature keratinocytes with low adhesion capacity (Adh-/+). We finally demonstrated that the progeny of Adh+++EGF-Rlow precursors after long-term expansion is still capable of generating a pluristratified epidermis in a model for skin reconstruction. In conclusion, after further characterizing the phenotype of primitive epidermal precursors, we demonstrated a new function of TGF-beta1, which is to promote undifferentiated keratinocyte amplification.
Overexpression of the HER2 (neu/c-erbB-2) oncogene frequently coincides with an aggressive clinical course of certain human adenocarcinomas. Expression and secretion of aberrant HER2 splice variants has been reported in various cell lines and tissues and can interfere with the oncogenic HER2 activity. Here we demonstrate, using two different approaches, that expression of a truncated 100 kDa HER2 variant which encodes the extracellular domain of HER2 (HER-ECD) inhibits growth factor-mediated tumour cell proliferation. A HER2-ECD cDNA encoding the truncated variant was overexpressed in MCF7 breast cancer cells. HER2-ECD overexpression decreased spontaneous proliferation of MCF7 cells as well as heregulin-mediated soft agar colony formation. Concomitantly, heregulin-induced phosphorylation of HER4 as well as downstream activation of p44/p42 MAP-kinases was decreased. To confirm these data, ribozymes were targeted to the 3'-untranslated region of the 2.3 kb HER2-ECD mRNA which is spontaneously expressed in MKN7 gastric cancer cells. HER2-ECD-targeted ribozymes downregulated HER2-ECD expression and enhanced EGF-mediated soft agar colony formation of MKN7 cells. In parallel, EGF-induced activation of p44/p42 MAP-kinases and activation of c-Fos expression were increased in ribozyme-transfected MKN7 cells. Finally, in RT-PCR we found a trend towards a progressive loss of 2.3 kb HER2-ECD mRNA expression in more advanced gastric tumours. These data show that the HER2-ECD variant inhibits growth factor-mediated tumour cell proliferation suggesting an important role during the progression of human cancer.
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