Expression of the cell surface receptor Fas is frequently lost or decreased during tumor progression in human colon carcinomas. The methylation status of a 583 bp CpG-rich region within the Fas promoter (À575 to +8) containing 28 CpG sites was determined in human colon carcinoma cell lines. In Caco2 (no Fas expression), 82-93% of CpG sites were methylated, whereas none were methylated in GC 3 /c1 (high Fas expression). In RKO (intermediate level of Fas), a single CpG site, located at -548, was 100% methylated. The inhibitor of methylation, 5-aza-2 0 -deoxycytidine (5-azadC), upregulated Fas expression in four of eight cell lines, and sensitized RKO cells to recombinant FasL-induced apoptosis. The p53-binding region in the first intron of the Fas gene was partially methylated in Caco2, and 5-azadC potentiated Ad-wtp53-induced upregulation of Fas expression. Methylation-specific PCR of the first intron detected partial methylation in four out of 10 colon carcinoma tumor samples in vivo. The data suggest that DNA hypermethylation is one mechanism that contributes to the downregulation of Fas expression and subsequent loss of sensitivity to Fas-induced apoptosis in colon carcinoma cells.

Dysregulation of the Notch‐pathway has been implicated in the pathogenesis of chronic lymphocytic leukaemia (B‐CLL). We characterized the mRNA expression of Notch pathway elements in circulating normal B‐ and B‐CLL cells, and compared expression profiles with clinical and prognostic data. Similar expression profiles were found in normal B‐cells and B‐CLL cells, however, most B‐CLL samples showed lower Hairy/Enhancer of Split‐1 expression than normal B‐cells, which suggests that the pathway is not over‐activated in B‐CLL. The expression of Notch‐pathway genes did not correlate with other prognostic factors of B‐CLL. The importance of Notch‐signalling in CLL cells in lymphatic tissue microenvironments remains to be determined.

Notch‐signalling has been implicated as a pathogenetic factor and a therapeutical target in T‐cell leukaemias and in some lymphomas of B‐cell origin. Our aim was to investigate the role of Notch‐signalling in apoptosis regulation in human non‐Hodgkin B‐cell lymphoma (B‐NHL) cell lines and in primary chronic lymhocytic leukaemia (CLL) cells using Delta‐like 4 (Dll4) ligand mediated Notch activation and gamma‐secretase inhibitor (GSI) mediated Notch inhibition in vitro. The potential cross‐talk of Notch with the transforming growth factor‐beta (TGFb) pathway in apoptosis induction was also explored, and the effect of GSI on drug‐induced apoptosis was assessed. Modulation of Notch‐signalling by itself did not change the rate of apoptosis in B‐NHL cell lines and in CLL cells. TGFb‐induced apoptosis was decreased – but not completely abolished – by GSI in TGFb‐sensitive cell lines, but resistance to the apoptotic effects of TGFb were not reversed by Notch activation or inhibition. Drug‐induced apoptosis was not modified by GSI. We identified Hairy/Enhancer of Split (HES)‐1 as a TGFb target gene in selected – TGFb‐sensitive – B‐NHL cell lines. TGFb‐induced HES‐1 was only partially Notch‐dependent in later phases. Apoptosis regulation by TGFb and GSI was not dependent on the transcriptional regulation of c‐myc. In conclusion, our data does not support a unifying role of Notch in regulating apoptosis in B‐NHL, but warns that gamma‐secretase inhibitors may actually counteract apoptosis in some cases.

No abstract

Several bifunctional nitrosoureido sugar alcohol derivatives were synthesized at the Institute for Drug Research in Budapest. For detailed studies two D,L-threitol derivatives have been selected (GYKI 13 324, GYKI 13 485). These compounds were less toxic and more effective than BCNU, the calculated therapeutic index values in a broad scale of transplantable, induced and heterotransplanted tumours were found to be more advantageous than with the referent compound. Both GYKI 13 324 and GYKI 13 485 exert their curative effect when given orally even in leukaemia types of tumours. Their effect on the blood picture of rats is moderate and of short duration.