Apoptotic activity of the atypical retinoids, CD437 and ST1926, in acute myeloid leukemia cells characterized by sensitivity and induced resistance: DNA damage, copy-number and expression studies.

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Retinoid related molecules (RRMs) are novel agents with tumor-selective apoptotic activity, an unusual mechanism of action and no-cross-resistance with other chemotherapeutics. CD437 and ST1926 are prototypic RRMs. ST1926 is more powerful and less toxic than CD437, and is currently undergoing phase I clinical trials. Although RRMs activate classic nuclear retinoic acid receptors, they also activate an as yet unknown second receptor-independent pathway, which is fundamental for the apoptotic activity. One of the earliest events triggered by RRMs in the tumor cell is a long-lasting increase in the levels of cytosolic calcium. Inhibition of the rise in calcium by chelating agents (BAPTA) or calcium blockers (nicardipine) suppresses or delays the apoptotic response. We recently developed a subline (NB4.437r) with selective resistance to RRMs (70-100 fold resistance index to CD437 and ST1926) from the acute promyelocytic leukemia cell line, NB4. In parental NB4 cells, apoptosis is preceded by a DNA damage response, which was evaluated by determination of histone H2AX phosphorylation and comet assays performed in alkaline conditions. These phenomena are dose and time dependent and are evident very early (30-60 min) and well before the appearance of any sign of apoptosis, such as annexin V positivity. Increases in H2AX phosphorylation is maximal during the S-phase of the cell cycle. This suggests that RRMs induce DNA double strand breaks during DNA synthesis and explains S-phase specificity in the apoptotic response of NB4 cells to RRMs. In NB4.437r cells, neither CD437 nor ST1926 induce DNA damage, supporting an important role of this event in the ensuing apoptotic process. In NB4 cells, DNA double strand breaks are completely suppressed by BAPTA and nicardipine. This indicates that the RRM-dependent calcium rise is instrumental in the generation of DNA double strand breaks. DNA damage may be the consequence of a direct or indirect genotoxic effect triggered by RRMs. To gain further insights into the molecular mechanisms responsible for the sensitivity/resistance to RRMs, NB4 and NB4.437r cells were studied for gene-copy-number and gene-expression, using CGH and GX microarrays consisting of 244,000 and 44,000 probes, respectively. The results obtained demonstrate that NB4.437r cells contain a specific 6-fold amplification of chromosome 1 band p32, which results in over-expression of more than 30 genes located in this DNA region. The data were confirmed and complemented by chromosomal FISH, which demonstrates the presence of a third copy of chromosome 1 carrying a multiple tandem amplification. We are currently using siRNA libraries targeting the genes present in the amplified segment to demonstrate the functional significance of these genes in RRM-induced DNA damage and apoptosis.