The prognostic relevance of flt3 and npm1 mutations on older patients treated intensively or non-intensively: a study of 1312 patients in the UK NCRI AML16 trial.

Although the prognostic impact of mutations of FLT3 and NPM1 have been extensively studied in younger patients with acute myeloid leukaemia, less is known in older patients whether treated intensively or non-intensively, or in the context of existing prognostic scores. In 1312 patients 16 and 21%, respectively had an FLT3 and NPM1 mutation. An FLT3 mutation did not affect remission rate in intensively or non-intensively treated patients but was associated with an inferior survival. All patients with an NPM1c mutation had a significantly higher remission rate irrespective of treatment approach but survival was not improved, overall, or in any genotype except as in younger patients, in the FLT3 WT NPM1c mutant subgroup. When incorporated into an established multi-parameter prognostic risk score, the molecular information provided additional prognostic definition in 11% of patients.

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 (CML), 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 evolution and cytogenetic instability in these cells alone. At 10(6) and 10(7) 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 10(5) and 10(4) dosage levels there were similar survival rates between mice injected with 32Db2-a2 or 32Db3-a2 while mice injected with 32Dp210 had a significantly shorter survival time. The study of this 32D cell line panel indicated that there were no overt differences in the biological properties conferred 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 panel of BCR-ABL expressing 32D cells provides a useful model for CML disease progression studies.

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.

Inhibition of mitochondrial function in HL60 cells is associated with an increased apoptosis and expression of CD14.

The myelomonocytic cell line HL60 can be induced by a variety of chemical agents to differentiation to either neutrophils or monocytes. Examination of gene expression, by differential display, in cells induced to monocytes with 1alpha,25-dihydroxyvitamin D(3) or neutrophils with all-trans retinoic acid (ATRA) identified a number of clones with altered patterns of expression over the period of differentiation. One of these clones was the mitochondrial gene NADH dehydrogenase subunit 4 (ND4) which showed a differential pattern of expression between the neutrophil and monocyte lineages. The potential of mitochondrial inhibitors to induce differentiation was investigated by treating the HL60 cells with either the NADH dehydrogenase inhibitor, Rotenone, the complex III inhibitor, Antimycin A, or the highly specific mitochondrial ATP-synthase inhibitor, Oligomycin. Although functional assays of differentiation did not produce any positive results, all the inhibitors resulted in a dramatic increase in CD14 expression at day 1, with CD38 markers not observed until day 3. The increased expression of CD14 was accompanied by a decrease in viability and all CD14 positive cells were also positive for Annexin V, a marker of apoptosis. These results suggest that inhibition of the components of the mitochondrial pathways may lead to the marking of some cells, via CD14, for cell death, whilst allowing commitment to differentiation to occur in the surviving population.

Identification of a retinoic acid responsive aldoketoreductase expressed in HL60 leukaemic cells.

Neutrophil and monocyte differentiation can be induced in HL60 leukaemia cells by all-trans-retinoic acid (ATRA) and 1alpha,25-dihydroxyvitamin D3 (D3), respectively, whose differentiating effects can be enhanced by exposure to 'anti-inflammatory agents' and steroids. We have provided evidence that this potentiation is via inhibition of the activity of an enzyme of the aldoketoreductase (AKR) family, but had failed to identify expression of known AKRs in HL60 cells. In this study, we have identified a previously unclassified aldoketoreductase family member (termed HAKR e) that is expressed in HL60 cells. HAKR e is dramatically and transiently up-regulated in HL60 cells within 24 h of exposure to ATRA, further supporting the proposition that a member(s) of this family of enzymes play(s) a role in controlling cell growth and/or differentiation.

Phorbol ester activation of protein kinase C inhibits CNP-stimulated cyclic GMP production in the mouse AtT-20 pituitary tumour cell line.

Preincubation of AtT-20 mouse pituitary tumour cells with the phorbol ester PMA resulted in a concentration-dependent inhibition of CNP-stimulated cyclic GMP production. The phorbol ester analogue 4 alpha phorbol had no inhibitory effect and 24 h preincubations with PMA resulted in a characteristic down-regulation of the response indicating that the inhibitory actions were mediated via the activation of protein kinase C. Forskolin in the presence of the phosphodiesterase inhibitor IBMX stimulated intracellular cyclic AMP concentrations by up to eight fold, but did not alter basal nor CNP-stimulated cyclic GMP production. These results indicate that CNP-stimulated guanylate cyclase activity associated with the GC-B natriuretic peptide receptor expressed in AtT-20 cells is inhibited by protein kinase C.

Characterization of natriuretic peptide receptor subtypes in the AtT-20 pituitary tumour cell line.

Receptors for the natriuretic peptide family have been characterized in the adrenocorticotrophic hormone (ACTH)-secreting AtT-20 pituitary tumour cell line. Northern blot analysis detected mRNA transcripts for the guanylate cyclase-linked GC-B receptor subtype. There was no evidence for the expression of either guanylate cyclase-linked GC-A receptor or atrial natriuretic peptide (ANP)-C (clearance) receptor mRNAs. Cyclic GMP production in AtT-20 cells was stimulated up to 200-fold by C-type natriuretic peptide (CNP), which was 10- and 20 times as effective as equivalent concentrations of brain natriuretic peptide and ANP respectively. Cyclic GMP dose-response curves to CNP failed to show any signs of saturation even at concentrations up to 30 microM, indicating a relatively low affinity of CNP for the GC-B receptor. Although CNP induced large stimulations in cyclic GMP production, specific binding of [125I-Tyr0]CNP could not be demonstrated in AtT-20 cells. The absence of specific binding with this radiolabelled analogue is possibly due to a reduced affinity for the GC-B receptor, as CNP analogues with N-terminal modifications such as [Tyr0]CNP and [127I-Tyr0]CNP exhibited reduced abilities to stimulate cyclic GMP production in these cells. Despite elevating cyclic GMP levels, CNP had no effect on basal or corticotrophin-releasing factor-stimulating ACTH release from the cells. These results show that the guanylate cyclase-coupled GC-B receptor is the only natriuretic peptide receptor subtype expressed in AtT-20 cells. Although CNP can markedly stimulate cyclic GMP production in these cells, there is incomplete expression of the normal natriuretic peptide-induced inhibitory pathway of ACTH secretion at some point distal to the production of cyclic GMP.

Atrial natriuretic peptide effects in AtT-20 pituitary tumour cells.

Whether atrial natriuretic peptide (ANP)-evoked inhibition of corticotrophin-releasing factor (CRF)-stimulated ACTH secretion was also manifest in ACTH secreting AtT-20 pituitary tumour cells was investigated. ANP stimulated increases in cGMP accumulation at concentrations of the peptide above 10(-8) M which indicates the presence of the ANP receptors on these cells. CRF stimulated a concentration-dependent increase in ACTH secretion from AtT-20 cells which was unaffected by ANP, 8-bromo-cGMP, or sodium nitroprusside (SNP). Calcium stimulated a concentration-dependent increase in ACTH secretion from electrically permeabilised cells which was unaffected by co-incubation with cGMP but potentiated by cAMP. These results reveal the presence of ANP receptors on AtT-20 cells but suggest that an incomplete expression of the stimulus-secretion coupling mechanisms for ANP, at some point after cGMP production, prevents the effects of natriuretic peptides upon ACTH secretion being manifest in these cells.