Jean-François Cajot

Overexpression of the p73 gene is a novel finding in high-risk B-cell chronic lymphocytic leukemia

The p73 protein shares structural and functional similarities with the tumour-suppressor p53, but its role in neoplastic transformation is unknown. Alternative splicing leads to the expression of at least nine p73 C-terminal mRNA splice variants (alpha, beta, gamma, delta, epsilon, zeta, eta, eta1, theta). In this survey, we analyse the expression of p73 by real-time quantitative RT-PCR, its known C-terminal variants with an RT-PCR-Southern technique and by Western blot in samples of 51 patients with B-CLL, normal B lymphocytes from eight individuals, and five haematopoetic cell lines. p73alpha protein expression positively correlated with higher risk B-CLL stages (P = 0.046). Total p73 mRNA expression was higher (P = 0.01) and p73alpha protein more frequently detected (P = 0.008) in B-CLL compared with normal CD19+-B-lymphocytes. p73 C-terminal mRNA variants were expressed both in B-CLL and in normal B-lymphocytes, but their expression was biased since the gamma (P = 0.041), the theta (P < 0.001), and the eta variant (P = 0.033) prevailed in normal B-lymphocytes. In summary, we conclude that the accumulation of p73, the expression pattern of particular p73 variants and its link to progression may play a distinct role in the molecular pathology B-CLL.

Differential expression of p73 splice variants and protein in benign and malignant ovarian tumours.

The p73 gene encodes a protein with substantial structural and functional similarities to the tumour‐suppressor p53. Alternative splicing of p73 mRNA leads to expression of 6 known RNA species and proteins (α, β, γ, δ, ϵ, ζ). We analysed the expression of these splice variants in ovarian adenocarcinoma by RT‐PCR followed by detection of amplicons with the Southern technique and by immunoblot in 32 malignant and benign epithelial ovarian tumour specimens and 3 ovarian adenocarcinoma cell lines (A2780, 2008, OVCAR‐3). p73α mRNA was expressed in all 17 ovarian cancer specimens, and 14 of 17 expressed at least 3 splice variants. In contrast, a different expression pattern was present in the ovarian adenomas: p73α was detected in 6 of 12 benign tumours, and only 1 adenoma expressed 3 splice variants. p73 protein was expressed in 9 of 16 ovarian cancer specimens, in all cell lines and in 1 of 3 borderline tumours. In contrast, none of 9 ovarian adenomas expressed detectable amounts of p73 protein. Expression of p73 mRNA and protein was not correlated with FIGO stage and histological grade, but we observed a significant correlation with over‐expression of p53 protein. In summary, epithelial ovarian cancers express a more complex p73 isoform pattern and higher levels of p73 mRNA and protein than ovarian adenomas. Int. J. Cancer 88:66–70, 2000.

The cyclin-dependent kinase inhibitors p18INK4c and p19INK4d are highly expressed in CD34+ progenitor and acute myeloid leukaemic cells but not in normal differentiated myeloid cells

Cyclin‐dependent kinase inhibitors (CKIs) are important for the differentiation of cells in various tissues. In acute myeloid leukaemia (AML) the cells accumulate at particular stages of myeloid maturation. We therefore analysed the expression pattern of different CKIs in fresh samples of AML patients and compared it with that in CD34+ progenitor and normal differentiated myeloid cells. Competitive RT‐PCR and Western analysis revealed a significantly higher expression of p18INK4c and p19INK4d in leukaemic and CD34+ progenitor cells than in granulocytes and monocytes. A different pattern was seen for p27Kip1 and p57Kip2 expression being low in leukaemic cells but high in normal immature and differentiated cells. No marked differences were found in p15INK4b and p21Cip1 mRNA expression between leukaemic and CD34+ progenitor or mature myeloid cells. Our findings therefore indicate that high expression of p18INK4c and p19INK4d in haemopoietic progenitor and leukaemic blast cells may contribute to the premature differentiation block seen in AML.

Different p16INK4a and p14ARF Expression Patterns in Acute Myeloid Leukaemia and Normal Blood Leukocytes

The p16INK4a gene is often disrupted or transcriptionally silenced by CpG island methylation in human cancers. However, in acute myeloid leukaemia (AML) alterations of the INK4a-ARF tumour suppressor locus are rarely found despite the noted variable p16INK4a mRNA and protein levels. The p14ARF, an alternative reading frame protein encoded from the same INK4a-ARF locus, is a potent tumour suppressor functionally linked to p53. There is little known regarding the role of p14ARF in primary human tumours. Therefore, we analysed the expression patterns of these two tumour suppressors in 37 cases of AML. The relative expression of p161NK4a and p14ARF mRNA in AML blasts, measured by a specific p16INKa/p14ARF multiplex RT-PCR, was significantly shifted towards p14ARF whereas relatively lower levels of p16INK4a were detected. Quantitative RT-PCR revealed significantly higher expression of both transcripts in AML blasts when compared to normal differentiated myeloid cells or CD34+ progenitor cells. Furthermore, a good correlation between p16INK4a protein and mRNA was observed, whereas no correlation was found with p14ARF. Our results suggest: a) increased levels of both p16INKa and p14ARF may participate in the pathogenesis of AML, b) that high p14ARF mRNA expression might influence p16INK4a transcription and c) that post-transcriptional regulatory mechanisms are important for p14ARF expression.

Modulation of the malignant phenotype with the urokinase-type plasminogen activator and the type I plasminogen activator inhibitor

Gene transfer techniques were utilized to evaluate the role of urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type 1 (PAI-1) in enhancing or preventing the expression of the invasive malignant phenotype, respectively. Mouse L-cell transfectants expressing human uPA or human PAI-1 as well as mouse B16 transfectants expressing mouse uPA or human PAI-1 were generated. These transfectants were tested using a variety of experimental methods including smooth muscle cell matrix solubilization in vitro, lung colony formation in vivo and co-cultures of antagonist-expressing cells in vitro. Results from these studies provide direct evidence for an enhancing role of uPA in malignant invasion and experimental metastasis and for a modulatory role of PAI-1 in tumor cell-mediated breakdown of extracellular matrices.