Most small cell lung cancer (SCLC) patients relapse within 12 months of starting combination chemotherapy plus radio-therapy, due to the development of acquired chemo-and radio-resistance. This phenomenon relates to the induction of tumour differentiation, resulting in apoptosis-resistant, morphologically variant (v-SCLC) cells, which lack the neuroendocrine expression of classic (c-) SCLC cells. In this study spontaneously adherent SCLC sublines were shown by differential gene expression analysis to provide an in vitro model of variant differentiation in SCLC, with down-regulation of neuroendocrine markers and up-regulation of epithelial differentiation markers cyclin D1, endothelin, the cell adhesion molecules CD 44 and integrin subunits a2, b3 and b4. The sensitivity of adherent SCLC sublines to etoposide, cyclophosphamide and gamma radiation was significantly diminished relative to parent suspension cell lines. Western blot analysis using phosphorylationspecific antibodies to Akt and MAP kinase revealed markedly elevated activation in adherent SCLC sublines, paralleled by increased levels of phosphorylated Bad protein and activated NF-kB. Subcultivation of the adherent sublines on uncoated surfaces reversed their adherent phenotype immediately and under these conditions Akt activity reverted to low levels. These results suggest that c-SCLC cells can differentiate spontaneously to v-SCLC and that the associated cellular adhesion may trigger Akt-dependent inhibition of apoptosis in SCLC cells, thus leading to acquired chemoand radio-resistance.
The tumor suppressor p16INK4a has functions beyond cell‐cycle control via cyclin‐dependent kinases. A coordinated remodeling of N‐ and O‐glycosylation, and an increase in the presentation of the endogenous lectin galectin‐1 sensing these changes on the surface of p16INK4a‐expressing pancreatic carcinoma cells (Capan‐1), lead to potent pro‐anoikis signals. We show that the p16INK4a‐dependent impact on growth‐regulatory lectins is not limited to galectin‐1, but also concerns galectin‐3. By monitoring its expression in relation to p16INK4a status, as well as running anoikis assays with galectin‐3 and cell transfectants with up‐ or downregulated lectin expression, a negative correlation between anoikis and the presence of this lectin was established. Nuclear run‐off and northern blotting experiments revealed an effect of the presence of p16INK4a on steady‐state levels of galectin‐3‐specific mRNA that differed from decreasing the transcriptional rate. On the cell surface, galectin‐3 interferes with galectin‐1, which initiates signaling toward its pro‐anoikis activity via caspase‐8 activation. The detected opposite effects of p16INK4a at the levels of growth‐regulatory galectins‐1 and ‐3 shift the status markedly towards the galectin‐1‐dependent pro‐anoikis activity. A previously undescribed orchestrated fine‐tuning of this effector system by a tumor suppressor is discovered.
Background and aim: The role of transforming growth factor β-1 (TGFβ-1) in neuroendocrine tumour biology is currently unknown. We therefore examined the expression and biological significance of TGFβ signalling components in neuroendocrine tumours (NETs) of the gastroenteropancreatic (GEP) tract. Methods: Expression of TGFβ-1 and its receptors, Smads and Smad regulated proteins, was examined in surgically resected NET specimens and human NET cell lines by immunohistochemistry, reverse transcriptase-polymerase chain reaction, immunoblotting, and ELISA. Activation of TGFβ-1 dependent promoters was tested by transactivation assays. Growth regulation was evaluated by cell numbers, soft agar assays, and cell cycle analysis using flow cytometry. The role of endogenous TGFβ was assessed by a TGFβ neutralising antibody and stable transfection of a dominant negative TGFβR II receptor construct. Results: Coexpression of TGFβ-1 and its receptors TGFβR I and TGFβR II was detected in 67% of human NETs and in all three NET cell lines examined. NET cell lines expressed the TGFβ signal transducers Smad 2, 3, and 4. In two of the three cell lines, TGFβ-1 treatment resulted in transactivation of a TGFβ responsive reporter construct as well as inhibition of c-myc and induction of p21 (WAF1) expression. TGFβ-1 inhibited anchorage dependent and independent growth in a time and dose dependent manner in TGFβ-1 responsive cell lines. TGFβ-1 mediated growth inhibition was due to G1 arrest without evidence of induction of apoptosis. Functional inactivation of endogenous TGFβ revealed the existence of an autocrine antiproliferative loop in NET cells. Conclusions: Neuroendocrine tumour cells of the gastroenteropancreatic tract are subject to paracrine and autocrine growth inhibition by TGFβ-1, which may account in part for the low proliferative index of this tumour entity.
Transitions from small cell (SCLC) to non-small cell lung cancer (NSCLC) cells have been documented both in vitro and in vivo and are thought to be an important step during tumor progression of human small cell lung cancer towards a treatment-resistant tumor state. We have screened NSCLC and SCLC cell lines for dierences in the composition of nuclear transcription factors using consensus oligonucleotide sequences (SRE, Ets, TRE, CRE, B-motif, GAS, E-box). We found NSCLC cells to exhibit signi®cantly higher AP-1 binding activity than SCLC cells consistent with the increased expression of CD44, an AP-1 target gene. To gain more insight into the molecular mechanisms underlying these dierences, we analysed SCLC cell lines (NCI-N592 and NCI-H69) which were phenotypically transformed into NSCLC-type cells by transfection with activated H-ras and c-myc oncogenes. In these cells, ras-induced transition is accompanied by a strong induction of AP-1-binding activity along with increased expression of CD44 mRNA and protein. When analysing the composition of the AP-1 complex in more detail and comparing ras-induced versus phorbol ester-induced changes, we found Fra-1 to be the major component induced in ras-transfected but not in phorbol-ester treated or non-treated parental SCLC cells. This ®nding is paralleled by the observation that among the various members of the Fos and Jun family analysed (c-Fos, FosB, Fra-1, Fra-2, c-Jun, JunD, JunB) fra-1 is the only gene to be exclusively expressed in NSCLC cells but not in cells of SCLC origin. Our data, thus, point to a histiotype-related mechanism of recruitment among AP-1 proteins which may have bearings on the fate of lung cancer development.
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