Expression of transforming growth factor beta (TGF beta) receptors and expression of TGF beta 1, TGF beta 2 and TGF beta 3 in human small cell lung cancer cell lines.

A panel of 21 small cell lung cancer cell (SCLC) lines were examined for the presence of Transforming growth factor beta receptors (TGF beta-r) and the expression of TGF beta mRNAs. By the radioreceptor assay we found high affinity receptors to be expressed in six cell lines. scatchard analysis of the binding data demonstrated that the cells bound between 4.5 and 27.5 fmol mg-1 protein with a KD ranging from 16 to 40 pM. TGF beta 1 binding to the receptors was confirmed by cross-linking TGF beta 1 to the TGF beta-r. Three classes of TGF beta-r were demonstrated, type I and type II receptors with M(r) = 65,000 and 90,000 and the betaglycan (type III) with M(r) = 280,000. Northern blotting showed expression of TGF beta 1 mRNA in ten, TGF beta 2 mRNA in two and TGF beta 3 mRNA in seven cell lines. Our results provide, for the first time, evidence that a large proportion of a broad panel of SCLC cell lines express TGF beta-receptors and also produce TGF beta mRNAs

In vitro invasion of small-cell lung cancer cell lines correlates with expression of epidermal growth factor receptor.

Formation of metastasis is a multistep process involving attachment to the basement membrane, local proteolysis and migration into surrounding tissues, lymph or bloodstream. In the present study, we have analysed the correlation between in vitro invasion and presence of the epidermal growth factor receptor (EGFR) in a panel of 21 small-cell lung cancer (SCLC) cell lines. We have previously reported that ten of these cell lines expressed EGFR protein detected by radioreceptor and affinity labelling assays. In 11 small-cell lung cancer (SCLC) cell lines, EGFR mRNA was detected by Northern blot analysis. In vitro invasion in a Boyden chamber assay was found in all EGFR-positive cell lines, whereas no invasion was detected in the EGFR-negative cell lines. Quantification of the in vitro invasion in 12 selected SCLC cell lines demonstrated that, in the EGFR-positive cell lines, between 5% and 16% of the cells added to the upper chamber were able to traverse the Matrigel membrane. Expression of several matrix metalloproteases (MMP), of tissue inhibitor of MMP (TIMP) and of cathepsin B was evaluated by immunoprecipitation, Western blot analysis and reverse transcriptase polymerase chain reaction (RT-PCR). However, in vitro invasive SCLC cell lines could not be distinguished from non-invasive cell lines based on the expression pattern of these molecules. In six SCLC cell lines, in vitro invasion was also determined in the presence of the EGFR-neutralizing monoclonal antibody mAb528. The addition of this antibody resulted in a significant reduction of the in vitro invasion in three selected EGFR-positive cell lines. Our results show that only EGFR-positive SCLC cell lines had the in vitro invasive phenotype, and it is therefore suggested that the EGFR might play an important role for the invasion potential of SCLC cell lines

Growth suppression by transforming growth factor beta 1 of human small-cell lung cancer cell lines is associated with expression of the type II receptor.

Nine human small-cell lung cancer cell lines were treated with transforming growth factor beta 1 (TGF-beta 1). Seven of the cell lines expressed receptors for transforming growth factor beta (TGF-beta-r) in different combinations between the three human subtypes I, II and III, and two were receptor negative. Growth suppression was induced by TGF-beta 1 exclusively in the five cell lines expressing the type II receptor. For the first time growth suppression by TGF-beta 1 of a cell line expressing the type II receptor without coexpression of the type I receptor is reported. No effect on growth was observed in two cell lines expressing only type III receptor and in TGF-beta-r negative cell lines. In two cell lines expressing all three receptor types, growth suppression was accompanied by morphological changes. To evaluate the possible involvement of the retinoblastoma protein (pRb) in mediating the growth-suppressive effect of TGF-beta 1, the expression of functional pRb, as characterised by nuclear localisation, was examined by immunocytochemistry. Nuclear association of pRb was only seen in two of the five TGF-beta 1-responsive cell lines. These results indicate that in SCLC pRb is not required for mediation of TGF-beta 1-induced growth suppression

Inactivation of the transforming growth factor β type II receptor in human small cell lung cancer cell lines

Transforming growth factor β (TGF-β) exerts a growth inhibitory effect on many cell types through binding to two types of receptors, the type I and II receptors. Resistance to TGF-β due to lack of type II receptor (RII) has been described in some cancer types including small cell lung cancer (SCLC). The purpose of this study was to examine the cause of absent RII expression in SCLC cell lines. Northern blot analysis showed that RII RNA expression was very weak in 16 of 21 cell lines. To investigate if the absence of RII transcript was due to mutations, we screened the poly-A tract for mutations, but no mutations were detected. Additional screening for mutations of the RII gene revealed a GG to TT base substitution in one cell line, which did not express RII. This mutation generates a stop codon resulting in predicted synthesis of a truncated RII of 219 amino acids. The nature of the mutation, which has not previously been observed in RII, has been linked to exposure to benzo[a]-pyrene, a component of cigarette smoke. Since RII has been mapped to chromosome 3p22 and nearby loci are often hypermethylated in SCLC, it was examined whether the lack of RII expression was due to hypermethylation. Southern blot analysis of the RII promoter did not show altered methylation patterns. The restriction endonuclease pattern of the RII gene was altered in two SCLC cell lines when digested with Sma 1. However, treatment with 5-aza-2′-deoxycytidine did not induce expression of RII mRNA. Our results indicate that in SCLC lack of RII mRNA is not commonly due to mutations and inactivation of RII transcription was not due to hypermethylation of the RII promoter or gene. Thus, these data show that in most cases of the SCLC cell lines, the RII gene and promoter is intact in spite of absent RII expression. However, the nature of the mutation found could suggest that it was caused by cigarette smoking. © 1999 Cancer Research Campaig

Proneoplastic effects of PGE2 mediated by EP4 receptor in colorectal cancer

<p>Abstract</p> <p>Background</p> <p>Prostaglandin E₂(PGE₂) is the major product of Cyclooxygenase-2 (COX-2) in colorectal cancer (CRC). We aimed to assess PGE₂cell surface receptors (EP 1–4) to examine the mechanisms by which PGE₂regulates tumour progression.</p> <p>Methods</p> <p>Gene expression studies were performed by quantitative RT-PCR. Cell cycle was analysed by flow cytometry with cell proliferation quantified by BrdU incorporation measured by enzyme immunoassay. Immunohistochemistry was employed for expression studies on formalin fixed paraffin embedded tumour tissue.</p> <p>Results</p> <p>EP4 was the most abundant subtype of PGE₂receptor in HT-29 and HCA7 cells (which show COX-2 dependent PGE₂generation) and was consistently the most abundant transcript in human colorectal tumours (n = 8) by qRT-PCR (ANOVA, p = 0.01). G0/G1 cell cycle arrest was observed in HT-29 cells treated with SC-236 5 μM (selective COX-2 inhibitor) for 24 hours (p = 0.02), an effect abrogated by co-incubation with PGE₂(1 μM). G0/G1 arrest was also seen with a specific EP4 receptor antagonist (EP4A, L-161982) (p = 0.01). Treatment of HT-29 cells with either SC-236 or EP4A caused reduction in intracellular cAMP (ANOVA, p = 0.01). Early induction in p21^WAF1/CIP1expression (by qRT-PCR) was seen with EP4A treatment (mean fold increase 4.4, p = 0.04) while other genes remained unchanged. Similar induction in p21^WAF1/CIP1was also seen with PD153025 (1 μM), an EGFR tyrosine kinase inhibitor, suggesting EGFR transactivation by EP4 as a potential mechanism. Additive inhibition of HCA7 proliferation was observed with the combination of SC-236 and neutralising antibody to amphiregulin (AR), a soluble EGFR ligand. Concordance in COX-2 and AR localisation in human colorectal tumours was noted.</p> <p>Conclusion</p> <p>COX-2 regulates cell cycle transition via EP4 receptor and altered p21^WAF1/CIP1expression. EGFR pathways appear important. Specific targeting of the EP4 receptor or downstream targets may offer a safer alternative to COX-2 inhibition in the chemoprevention of CRC.</p

Displayed correlation between gene expression profiles and submicroscopic alterations in response to cetuximab, gefitinib and EGF in human colon cancer cell lines

Background: EGFR is frequently overexpressed in colon cancer. We characterized HT-29 and Caco-2, human colon cancer cell lines, untreated and treated with cetuximab or gefitinib alone and in combination with EGF. Methods: Cell growth was determined using a variation on the MTT assay. Cell-cycle analysis was conducted by flow cytometry. Immunohistochemistry was performed to evaluate EGFR expression and scanning electron microscopy (SEM) evidenced the ultrastructural morphology. Gene expression profiling was performed using hybridization of the microarray Ocimum Pan Human 40 K array A. Results: Caco-2 and HT-29 were respectively 66.25 and 59.24 % in G0/G1. They maintained this level of cell cycle distribution after treatment, suggesting a predominantly differentiated state. Treatment of Caco-2 with EGF or the two EGFR inhibitors produced a significant reduction in their viability. SEM clearly showed morphological cellular transformations in the direction of cellular death in both cell lines treated with EGFR inhibitors. HT-29 and Caco-2 displayed an important reduction of the microvilli (which also lose their erect position in Caco-2), possibly invalidating microvilli absorption function. HT-29 treated with cetuximab lost their boundary contacts and showed filipodi; when treated with gefitinib, they showed some vesicles: generally membrane reshaping is evident. Both cell lines showed a similar behavior in terms of on/off switched genes upon treatment with cetuximab. The gefitinib global gene expression pattern was different for the 2 cell lines; gefitinib treatment induced more changes, but directly correlated with EGF treatment. In cetuximab or gefitinib plus EGF treatments there was possible summation of the morphological effects: cells seemed more weakly affected by the transformation towards apoptosis. The genes appeared to be less stimulated than for single drug cases. Conclusion: This is the first study to have systematically investigated the effect of cetuximab or gefitinib, alone and in combination with EGF, on human colon cancer cell lines. The EGFR inhibitors have a weaker effect in the presence of EGF that binds EGFR. Cetuximab treatment showed an expression pattern that inversely correlates with EGF treatment. We found interesting cytomorphological features closely relating to gene expression profile. Both drugs have an effect on differentiation towards cellular death