Inactivation of the tumour suppressor p53 is the most common defect in cancer cells. p53 is a sequence specific transcription factor that is activated in response to various forms of genotoxic stress to induce cell cycle arrest and apoptosis. Induction of p53 is subjected to complex and strict control through several pathways, as it will often determine cellular fate. The p73 protein shares strong structural and functional similarities with p53 such as the potential to activate p53 responsive genes and the ability to induce apoptosis. In addition to alternative splicing at the carboxyl terminus which yields several p73 isoforms, a p73 variant lacking the Nterminal transactivation domain (DNp73) was described in mice. In this study, we report the cloning and characterisation of the human DNp73 isoforms, their regulation by p53 and their possible role in carcinogenesis. As in mice, human DNp73 lacks the transactivation domain and starts with an alternative exon (exon 3'). Its expression is driven by a second promoter located in a genomic region upstream of this exon, supporting the idea of two independently regulated proteins, derived from the same gene. As anticipated, DNp73 is capable of regulating TAp73 and p53 function since it is able to block their transactivation activity and their ability to induce apoptosis. Interestingly, expression of the DNp73 is strongly upregulated by the TA isoforms and by p53, thus creating a feedback loop that tightly regulates the function of TAp73 and more importantly of p53. The regulation of DNp73 is exerted through a p53 responsive element located on the DN promoter. Expression of DNp73 not only regulates the function of p53 and TAp73 but also shuts off its own expression, once again finely regulating the whole system. Our data also suggest that increased expression of DNp73, functionally inactivating p53, could be involved in tumorogenesis. An extensive analysis of the expression pattern of DNp73 in primary tumours would clarify this issue. Cell Death and Differentiation (2001) 8, 1213 ± 1223.

We have cloned two novel, alternatively spliced messages of human cyclin D-binding Myb-like protein (hDMP1). The known, full-length protein has been named hDMP1␣ and the new isoforms, hDMP1␤ and hDMP1␥. The hDMP1␣, -␤, and -␥ splice variants have unique expression patterns in normal hematopoietic cells; hDMP1␤ mRNA transcripts are strongly expressed in quiescent CD34؉ cells and freshly isolated peripheral blood leukocytes, as compared with hDMP1␣. In contrast, activated T-cells and developing myeloid cells, macrophages, and granulocytes express low levels of hDMP1␤ transcripts, and hDMP1␥ is ubiquitously and weakly expressed. Mouse Dmp1 has been shown to activate CD13/aminopeptidase N (APN) and p19 ARF gene expression via binding to canonical DNA recognition sites in the respective promoters. Assessment of CD13/APN promoter responsiveness demonstrated that hDMP1␣ but not hDMP1␤ and -␥, is a transcriptional activator. Furthermore, hDMP1␤ was found to inhibit the CD13/ APN promoter transactivation ability of hDMP1␣. Stable, ectopic expression of hDMP1␤ and, to a lesser extent hDMP1␥, reduced endogenous cell surface levels of CD13/APN in U937 cells. Moreover, stable, ectopic expression of hDMP1␤ altered phorbol 12-myristate 13-acetate-induced terminal differentiation of U937 cells to macrophages and resulted in maintenance of proliferation. These results demonstrate that hDMP1␤ antagonizes hDMP1␣ activity and suggest that cellular functions of hDMP1 may be regulated by cellular hDMP1 isoform levels.

The CDKN2 locus expresses two di erent mRNA transcripts, designated a and b. The protein product of the a transcript is the cell cycle inhibitor and tumour suppressor p16INK4a. The b transcript is translated in an alternate reading frame (ARF) and in humans encodes a 15 kDa protein (p19ARF). Immunohistochemical and Western analysis of p16INK4a has shown that the protein is downregulated in a signi®cant number of tumours, but less is known on the expression of the p19ARF. We have examined the expression of p16INK4a and p19ARF in resectable non-small cell lung cancer (NSCLC) by immunostaining (n=49) and multiplex RT ± PCR (n=28). In order to investigate the mechanism responsible for p16INK4a downregulation, exon 1a methylation was analysed in a PCR-based assay. Of 49 tumours examined by immunostaining, 24 and 20 tumours expressed p16INK4a and p19ARF at nil to low levels, respectively. p19ARF was localized primarily to the nuclei of tumour cells, but was also seen to varying degrees in nuclei of lymphocytes, chondrocytes, ®broblasts, and epithelial cells. No tumour with normal p16INK4a had decreased p19ARF expression. Among 16 tumours with nil to low p16INK4a expression, 11 tumours exhibited full methylation of at least one site within exon 1a and these tumours showed normal p19ARF expression. In contrast, no methylation of exon 1a was observed in ®ve tumours which also lacked p19ARF. In normal lung, p16INK4a and p19ARF were not expressed at detectable levels, the multiplex RT ± PCR results were balanced, and sites within exon 1a were strongly methylated. In tumours, imbalanced multiplex RT ± PCR data (p16INK4a5p19ARF) predicted methylation of exon 1a (P=0.0006) as well as downregulation of p16INK4a. p19ARF downregulation was inversely correlated with p53 overexpression (P=0.025), whilst negative immunostaining for p16INK4a was inversely correlated with pRb downregulation (P=0.003) and directly correlated with p53 overexpression as assessed by immunostaining (P=0.015). Our results show that: (1) p16INK4a and p19ARF expression are altered in almost half of resectable NSCLC; (2) methylation within exon 1a is a frequent, but not the only mechanism of p16INK4a downregulation; and that (3) the inverse association of p19ARF and p53 alteration is consistent with a linked pathway.

Early allogeneic hematopoietic stem cell transplantation (HSCT) has been proposed as primary treatment modality for patients with chronic myeloid leukemia (CML). This concept has been challenged by transplantation mortality and improved drug therapy. In a randomized study, primary HSCT and best available drug treatment (IFN based) were compared in newly diagnosed chronic phase CML patients. Assignment to treatment strategy was by genetic randomization according to availability of a matched related donor. Evaluation followed the intention-to-treat principle. Six hundred and twenty one patients with chronic phase CML were stratified for eligibility for HSCT. Three hundred and fifty four patients (62% male; median age, 40 years; range, 11-59 years) were eligible and randomized. One hundred and thirty five patients (38%) had a matched related donor, of whom 123 (91%) received a transplant within a median of 10 months (range, 2-106 months) from diagnosis. Two hundred and nineteen patients (62%) had no related donor and received best available drug treatment.

Summary Acute promyelocytic leukaemia (APL) patients are successfully treated with all‐trans retinoic acid (ATRA). However, concurrent chemotherapy is still necessary and less toxic therapeutic approaches are needed. Earlier studies suggested that in haematopoietic neoplasms, the green tea polyphenol epigallocatechin‐3‐gallate (EGCG) induces cell death without adversely affecting healthy cells. We aimed at deciphering the molecular mechanism of EGCG‐induced cell death in acute myeloid leukaemia (AML). A significant increase of death‐associated protein kinase 2 (DAPK2) levels was found in AML cells upon EGCG treatment paralleled by increased cell death that was significantly reduced upon silencing of DAPK2. Moreover, combined ATRA and EGCG treatment resulted in cooperative DAPK2 induction and potentiated differentiation. EGCG toxicity of primary AML blasts correlated with 67 kDa laminin receptor (67LR) expression. Pretreatment of AML cells with ATRA, causing downregulation of 67LR, rendered these cells resistant to EGCG‐mediated cell death. In summary, it was found that (i) DAPK2 is essential for EGCG‐induced cell death in AML cells, (ii) ATRA and EGCG cotreatment significantly boosted neutrophil differentiation, and 67LR expression correlates with susceptibility of AML cells to EGCG. We thus suggest that EGCG, by selectively targeting leukaemic cells, may improve differentiation therapies for APL and chemotherapy for other AML subtypes.