Malignant mesothelioma, clinical, diagnostic and cell biological investigations

The purpose of this study was to improve the diagnosis of human malignant mesothelioma on the one hand and to investigate the growth regulation and transformation of normal and malignant mesothelial cells on the other hand. In this thesis improvement of diagnosis was approached by the search for specific tumor markers. Therefore malignant mesotheliomas were analysed for their chromosomal aberrations and the expression of specific membrane antigen markers. For the investigation of the growth regulation in normal and malignant mesothelial cells a panel of in vitro growing malignant mesothelioma cell lines was established. These cell lines were studied for expression of growth factors and their receptors, with emphasis on the platelet-derived growth factor and its receptor. Evidence has been obtained that platelet-derived growth factor (PDGF) may play a role in the malignant transformation of mesothelial cells

Splicing of the platelet‐derived‐growth‐factor A‐chain mRNA in human malignant mesothelioma cell lines and regulation of its expression

Platelet‐derived‐growth‐factor (PDGF) A‐chain transcripts differing in the presence or absence of an alternative exon‐derived sequence have been described. In some publications, the presence of PDGF A‐chain transcripts with this exon‐6‐derived sequence was suggested to be tumour specific. However, in this paper it was shown by reverse‐transcription polymerase‐chain‐reaction (PCR) analysis that both normal mesothelial cells and malignant mesothelioma cell lines predominantly express the PDGF A‐chain transcript without the exon‐6‐derived sequence. This sequence encodes a cell‐retention signal, which means that the PDGF A‐chain protein is most likely to be secreted by both cell types. In cultured normal mesothelial cells, the secreted PDGF A‐chain protein might be involved in autocrine growth stimulation via PDGF α receptors. However, human malignant mesothelioma cell lines only possess PDGF β receptors. If this also holds true in vivo, the PDGF A‐chain protein produced and secreted by malignant mesothelial cells might have a paracrine function. In a previous paper, we described elevated expression of the PDGF A‐chain transcript in human malignant mesothelioma cell lines, compared to normal mesothelial cells. In this paper, the possible reason for this elevation was studied. First, alterations at the genomic level were considered, but cytogenetic and Southern‐blot analysis revealed neither consistent chromosomal aberrations, amplification nor structural rearrangement of the PDGF A‐chain gene in the malignant cells. Possible differences in transcription rate of the PDGF A‐chain gene, and stability of the transcript between normal and malignant cells, were therefore studied. The presence of a protein‐synthesis inhibitor, cycloheximide, in the culture medium did not significantly influence the PDGF A‐chain mRNA level in normal mesothelial and malignant mesothelioma cell lines. Furthermore, nuclear run‐off analysis showed that nuclear PDGF A‐chain mRNA levels varied in both cell types to the same extent as the levels observed in Northern blots. Taken together, this suggests that increased transcription is the most probable mechanism for the elevated mRNA level of the PDGF A‐chain gene in human malignant mesothelioma cell lines.</p

Splicing of the platelet‐derived‐growth‐factor A‐chain mRNA in human malignant mesothelioma cell lines and regulation of its expression

Platelet‐derived‐growth‐factor (PDGF) A‐chain transcripts differing in the presence or absence of an alternative exon‐derived sequence have been described. In some publications, the presence of PDGF A‐chain transcripts with this exon‐6‐derived sequence was suggested to be tumour specific. However, in this paper it was shown by reverse‐transcription polymerase‐chain‐reaction (PCR) analysis that both normal mesothelial cells and malignant mesothelioma cell lines predominantly express the PDGF A‐chain transcript without the exon‐6‐derived sequence. This sequence encodes a cell‐retention signal, which means that the PDGF A‐chain protein is most likely to be secreted by both cell types. In cultured normal mesothelial cells, the secreted PDGF A‐chain protein might be involved in autocrine growth stimulation via PDGF α receptors. However, human malignant mesothelioma cell lines only possess PDGF β receptors. If this also holds true in vivo, the PDGF A‐chain protein produced and secreted by malignant mesothelial cells might have a paracrine function. In a previous paper, we described elevated expression of the PDGF A‐chain transcript in human malignant mesothelioma cell lines, compared to normal mesothelial cells. In this paper, the possible reason for this elevation was studied. First, alterations at the genomic level were considered, but cytogenetic and Southern‐blot analysis revealed neither consistent chromosomal aberrations, amplification nor structural rearrangement of the PDGF A‐chain gene in the malignant cells. Possible differences in transcription rate of the PDGF A‐chain gene, and stability of the transcript between normal and malignant cells, were therefore studied. The presence of a protein‐synthesis inhibitor, cycloheximide, in the culture medium did not significantly influence the PDGF A‐chain mRNA level in normal mesothelial and malignant mesothelioma cell lines. Furthermore, nuclear run‐off analysis showed that nuclear PDGF A‐chain mRNA levels varied in both cell types to the same extent as the levels observed in Northern blots. Taken together, this suggests that increased transcription is the most probable mechanism for the elevated mRNA level of the PDGF A‐chain gene in human malignant mesothelioma cell lines.</p

Regulation of differential expression of platelet-derived growth factor α-and β-receptor mRNA in normal and malignant human mesothelial cell lines

In earlier studies we showed that the expression patterns of platelet-derived growth factor (PDGF) α- and β-receptors differ between normal and malignant mesothelial cell lines. Normal mesothelial cells predominantly express PDGF α-receptor mRNA and protein, whereas most malignant mesothelioma cell lines produce PDGF P-receptor mRNA and protein. In this paper we studied regulation of this differential PDGF receptor mRNA expression. Such an analysis is of importance in view of the suggested PDGF autocrine activity involving the PDGF β-receptor in mesothelioma cells. The results obtained in this study demonstrate that malignant mesothelioma cell lines are not only capable of PDGF β-receptor transcription but of α-receptor transcription as well, as evidenced from run off analysis and RT-PCR using α-receptor specific primers. However, the fact that PDGF α-receptor mRNA could not be detected by Northern blot analysis, even after cycloheximide treatment, suggests a difference in steady-state PDGF α-receptor mRNA expression levels between normal and malignant mesothelial cell lines, which is likely to be caused by a post-transcriptional mechanism. In normal mesothelial cells a half-life of more than 6 h was observed for PDGF α-receptor mRNA. In the majority of malignant mesothelioma cell lines clear PDGF β-receptor mRNA expression was seen. The half-life of the PDGF β-receptor transcript was at least 6 h in these cells. In contrast, hardly any PDGF β-receptor transcription was observed in run off assays in normal mesothelial cells, suggesting that differences in β-receptor transcriptional initiation most probably account for the inability to clearly detect PDGF β-receptor transcripts in these cells. Transforming growth factor β-1 (TGF-β1), which is being produced in active form by mesothelial cells was evaluated for its potential role in regulation of the differential PDGF receptor expression in these cells. Stimulation with TGF-β1 revealed decreased PDGF α-receptor mRNA expression in normal mesothelial cells. The effect on PDGF β-receptor mRNA in the malignant mesothelioma cell lines was variable. Although the TGF-β1 effect cannot entirely explain the differential PDGF receptor expression pattern, TGF-β1 may nevertheless play a role in downregulation of an (already) low PDGF α-receptor mRNA level in malignant mesothelioma cell lines.</p

Regulation of differential expression of platelet-derived growth factor α-and β-receptor mRNA in normal and malignant human mesothelial cell lines

In earlier studies we showed that the expression patterns of platelet-derived growth factor (PDGF) α- and β-receptors differ between normal and malignant mesothelial cell lines. Normal mesothelial cells predominantly express PDGF α-receptor mRNA and protein, whereas most malignant mesothelioma cell lines produce PDGF P-receptor mRNA and protein. In this paper we studied regulation of this differential PDGF receptor mRNA expression. Such an analysis is of importance in view of the suggested PDGF autocrine activity involving the PDGF β-receptor in mesothelioma cells. The results obtained in this study demonstrate that malignant mesothelioma cell lines are not only capable of PDGF β-receptor transcription but of α-receptor transcription as well, as evidenced from run off analysis and RT-PCR using α-receptor specific primers. However, the fact that PDGF α-receptor mRNA could not be detected by Northern blot analysis, even after cycloheximide treatment, suggests a difference in steady-state PDGF α-receptor mRNA expression levels between normal and malignant mesothelial cell lines, which is likely to be caused by a post-transcriptional mechanism. In normal mesothelial cells a half-life of more than 6 h was observed for PDGF α-receptor mRNA. In the majority of malignant mesothelioma cell lines clear PDGF β-receptor mRNA expression was seen. The half-life of the PDGF β-receptor transcript was at least 6 h in these cells. In contrast, hardly any PDGF β-receptor transcription was observed in run off assays in normal mesothelial cells, suggesting that differences in β-receptor transcriptional initiation most probably account for the inability to clearly detect PDGF β-receptor transcripts in these cells. Transforming growth factor β-1 (TGF-β1), which is being produced in active form by mesothelial cells was evaluated for its potential role in regulation of the differential PDGF receptor expression in these cells. Stimulation with TGF-β1 revealed decreased PDGF α-receptor mRNA expression in normal mesothelial cells. The effect on PDGF β-receptor mRNA in the malignant mesothelioma cell lines was variable. Although the TGF-β1 effect cannot entirely explain the differential PDGF receptor expression pattern, TGF-β1 may nevertheless play a role in downregulation of an (already) low PDGF α-receptor mRNA level in malignant mesothelioma cell lines.</p