Cell morphology showed a HOXB1 dependent increment in the number of terminally differentiated monocytes paralleled by a reduced amount of blast cells at day 7. Trying to understand the HOXB1 based mechanisms in inducing apoptosis and enhancing differentiation, we compared the differentiation level of HL60/HOXB1 vs control vector in presence or not of the caspase inhibitor z VAD and 1% of serum. Firstly, either in control conditions we confirmed the capability of HOXB1 to induce a cer tain degree of maturation. Indeed, up to day 6 of cell culture, HL60/LXSN only included undif ferentiated blasts, whereas approximately 40% of inter mediate differentiated cells were detectable in HOXB1 expressing HL60. The percentage of CD11b and G CSFR positive cells was increased from 31 to 66% and from 21 to 37% in LXSN vs HOXB1 transduced cells, respectively.

As supported in terms of microscopic analyses and CD11b cell surface marker, the presence of z VAD appeared to slightly interfere with the direct HOXB1 action. Conversely, the HOXB1 related differences, visible in ATRA treated cells, were maintained by the combination with z VAD, thus indi cating that HOXB1 induced sensitivity to ATRA is maintained blocking apoptosis. In these experiments the addition of z VAD seemed to be even more effective on cell differentiation, possibly through an accumulation of mature cells otherwise addressed to death. Expression analysis of HOXB1 regulated genes In order to gain insight in the molecular mechanisms underlying HOXB1 effects in the leukemic phenotype, we investigated genes differentially expressed in HOXB1 negative vs HOXB1 positive HL60 cells by probing an Atlas Human Cancer cDNA macroarray.

The expression level of some selected genes was confirmed by Real time RT PCR. Interestingly, among the differentially expressed genes, we found mol ecules that could directly explain the reduced ma lignancy of HOXB1 transduced cells. Some tumour promoting genes, related to cell growth and survival, like the early growth response 1, the fatty acid synthase and the mouse double minute 2 homo log, resulted in fact strongly down regulated, whereas pro apoptotic or tumor suppressor genes, as the caspase2, the pro grammed cell death 10, the non metastatic cells 1 protein, and the secreted protein acidic and rich in cysteine were up regulated.

HOXB1 promoter results methylated in HL60 To investigate the possible mechanisms underlying HOXB1 downregulation in leukemic cells, we compared the methylation status of the CpG island present on HOXB1 promoter in HL60 and in normal monocytes and granulocytes from peripheral blood. As shown by three separate experiments, the hypermethylated fraction of the HOXB1 Drug_discovery CpG island was significantly higher in HL60 respect to normal monocytes and granulocytes.