V. Walsh

Abnormalities of adherent layers grown from bone marrow of patients with myelodysplasia.

Myelodysplastic syndromes (MDS) are characterized by a clonal disorder of haemopoiesis with defective growth in vitro. The long‐term culture system was used to examine aspects of stromal function in MDS patients. Primary long‐term cultures of MDS bone marrow showed poor myelopoiesis with progenitors being detected for a median 3·5 weeks (n = 12) compared with 18 weeks in cultures of normal marrow (n = 10; P < 0·0001). The haemopoietic function of adherent layers was assessed in secondary co‐cultures seeded with 5 × 106 cord blood mononuclear cells on irradiated normal (n = 27; aged 38–82 years) or MDS (n = 32; aged 41–86 years) adherent layers (> 60% confluent). The median myeloid progenitor number/cord blood co‐culture was 135 in 5‐week‐old cultures with normal adherent layers and 22 in those with MDS layers (P < 0·0001). Myeloid colonies were detectable for a median 11 weeks with normal adherent layers and 6 weeks with MDS adherent layers (P < 0·0001); erythroid colonies were detectable for 7 weeks (normal) compared with 5 weeks (MDS) (P < 0·01). The differences in granulocyte‐macrophage colony forming unit (CFU‐GM) generation were not related to patient age. Cells from adherent layers of at least half of the primary normal (n = 48) and MDS (n = 26) long‐term cultures expressed cytokines [interleukin (IL)‐3, IL‐1β, thrombopoietin (Tpo) and erythropoietin (Epo)] and receptors for retinoic acid (RARα) [IL‐2, IL‐3, macrophage colony stimulating factor (M‐CSF) (Fms) and Tpo (Mpl)]. Only IL‐1β expression was reduced in week‐5 MDS cultures compared with those from normal marrows (P < 0·05). There was also a highly significant decline in IL‐1β expression in normal (but not MDS) adherent layers between week 5 and week 10. Thus, the adherent layers in cultures grown from MDS patients were haemopoietically defective and showed abnormal IL‐1β expression.

Heat shock protein 90 inhibition is cytotoxic to primary AML cells expressing mutant FLT3 and results in altered downstream signalling

Activating mutations of the FMS‐like tyrosine kinase 3 gene (FLT3) occur in approximately one‐third of patients with acute myeloid leukaemia (AML) and predict for a poor outcome. Heat shock protein 90 (Hsp90) is a molecular chaperone that is frequently used by cancer cells to stabilise mutant oncoproteins. Mutant FLT3 is chaperoned by Hsp90 in primary AML blasts whereas unmutated FLT3 is not, making Hsp90 inhibitors potentially useful therapeutically. The present study showed that inhibition of Hsp90 by 17‐allylamino‐17‐demethoxygeldanamycin (17‐AAG) was cytotoxic to primary AML cells expressing mutant FLT3. Inhibition of Hsp90 results in altered downstream signalling effects in primary AML cells with disruption of Janus kinase–signal transducer and activator of transcription (JAK‐STAT), mitogen‐activated protein kinase and phosphatidylinositol 3/AKT signalling pathways. Co‐treatment of blasts with 17‐AAG and cytarabine resulted in a synergistic or additive effect in approximately 50% of AML cases tested. Our results confirm that Hsp90 is a valid molecular target in the therapy of AML. Inhibition of Hsp90 in parallel with conventional AML therapies may have particular benefit in those patients with the poor prognostic FLT3 mutant disease.

High FUS/TLS expression in acute myeloid leukaemia samples

Retinoic acid has the ability to induce differentiation in some myeloid leukaemia cell lines and has been used to induce remission in acute promyelocytic leukaemia patients. We have analysed changes in gene expression, by differential display, in HL60 cells exposed to all‐trans retinoic acid (ATRA) for only 1 h. Only about 0.4% of the genes examined by this technique showed changes in expression level, and all four of the gene fragments identified were downregulated during the short 1 h exposure. Two of the fragments were novel, a third was MYC and the fourth was the FUS proto‐oncogene. Northern analysis showed that FUS was downregulated within 1 h only during induced neutrophil differentiation but not at all during induced monocyte differentiation. Unlike the sensitive cell lines, ATRA‐resistant cell lines did not show a downregulation of FUS over a 24 h period of exposure to ATRA. Using a semiquantitative PCR analysis, no difference in FUS levels was observed between ATRA‐sensitive and ‐resistant cell lines. A similar analysis was carried out on primary acute myeloid leukaemia (AML), peripheral stem cell harvests (PBSC) and cord blood samples. The PBSC and cord blood samples had FUS levels that were similar or generally less than the cell lines. However, much higher levels were seen in 63% of the AML samples examined. The data presented are consistent with previous reports for a role for FUS in the promotion and maintenance of cellular proliferation.

An in vivo and in vitro comparison of the effects of b2-a2 and b3-a2 p210BCR-ABL splice variants on murine 32D cells.

The Philadelphia (Ph) chromosome, a characteristic cytogenetic marker of chronic myeloid leukaemia (CMJL), is caused by a reciprocal translocation juxtaposing the 3′ region of the ABL gene onto the 5′ region of the BCR gene. Due to conservation of the reading frame, but depending on the site of the breakpoint in the BCR gene, two alternatively spliced variants of the p210BCR-ABL mRNA (known as b2-a2 and b3-a2) are produced. To investigate whether there are any biological differences between these splice variants we have transfected the b3-a2 or b2-a2 cDNA into a murine myeloid cell line, 32D. We have also included the previously prepared 32Dp210 cell line (which expresses the b3-a2 transcript) in all of our comparisons. RT-PCR analysis indicated that transcription levels were comparable between the variants. Morphological examination of the cells expressing either of the BCR-ABL transcripts indicated that these cells were more mature with increased cytoplasm:nuclear ratios compared to the 32D parental and 32Dneo vector control cells. However, the 32Dp210 cells had a very different appearance from the other panel members and flow karyotyping indicated a clonal ewolution and cytogenetic instability in these cells alone. At 106 and 107 cell doses all 32D cells; expressing BCR-ABL caused ill health and tissue infiltration in SCID mice with such rapidity that statistical analysis was not informative. However, at the 105 and 104 dosage levels there were similar survival rates between mice injected with 32Db2-a2 or 32Db3-a2 while mice injected with 32Dp210 had a significantly shorten-survival time. The study of this 32D cell line panel indicated that there were no overt differences in the biological properties conferned by the b3-a2 or b2-a2 transcripts to the 32D cells although these transcripts were able to confer in vitro and in vivo biological effects. This punel of BCR-ABL expressing 32D cells provides a useful model for CML disease progression studies.