Hiromichi Matsushita

All-trans and 9-cis retinoic acid enhance 1,25-dihydroxyvitamin D3-induced monocytic differentiation of U937 cells

Retinoic acid (RA) and 1,25-dihydroxyvitamin D3 (D3) are well known for inducing differentiation in many leukemic cell lines. The nuclear signalling pathways of RA and D3 are mediated through their cognate receptors, the retinoic acid receptor (RAR) and vitamin D3 receptor (VDR), respectively. Retinoid X receptor (RXR) is an auxiliary factor that forms a heterodimer with RAR and VDR, enabling their efficient transcriptional activation. 9-cis RA, a high-affinity ligand for RXR, greatly enhanced D3-induced CD14 expression in U937 cells, while RA alone did not induce CD14 expression. 9-cis RA also resulted in morphological changes of U937 cells to macrophage-like cells when combined with D3, while RA alone resulted in granulocyte-like cells. RA and D3 together enhanced c-fms expression, phagocytic activity, and acted synergistically to promote nitroblue tetrazolium reduction activity and inhibit proliferation. Northern analysis showed that U937 cells constitutively expressed RAR-alpha, VDR and RXR-alpha mRNAs. RA or D3 alone or in combination did not affect RAR-alpha and VDR expression, while 9-cis RA and 9-cis RA plus all-trans RA significantly reduced RXR-alpha expression. Interestingly, D3 could restore the down-regulation of RXR-alpha mRNA by 9-cis RA. These findings suggest that there is crossover of the nuclear signalling pathways of RA and D3. This may have clinical implications in that RA and D3 may be used in combination for differentiation-inducing therapy in acute myelogenous leukemia and myelodysplastic syndrome.

Serial quantification of minimal residual disease of t (8 ; 21) acute myelogenous leukaemia with RT-competitive PCR assay

The chromosomal translocation (8;21)(q22;q22) in the AML M2 subtype according to the FAB classification, results in the production of a novel fusion gene AML1/ETO. The chimaeric AML1/ETO transcript is useful for the detection of minimal residual disease (MRD). Recently, several studies on the detection of AML1/ETO transcripts in t(8;21) AML have been reported. However, the clinical significance of a small number of AML1/ETO transcripts by a reverse transcription–polymerase chain reaction (RT‐PCR) remains to be elucidated. We have developed a novel quantitative RT‐competitive PCR assay and evaluated the clinical usefulness of this method by the monitoring of MRD in eight patients with t(8;21) AML. In four patients in first continuous complete remission (CR) the value of MRD was always <0.1 fg of the competitor dose throughout their courses, whereas in four relapsed patients there was an increase in the value of MRD to <0.1 fg of the competitor dose before cytogenetic relapse. We conclude that the detection of the presence of cells with AML1/ETO fusion transcripts by our RT‐competitive PCR assay may be useful to monitor disease progression and to predict subsequent relapse.

A novel retinoic acid receptor (RAR)-selective antagonist inhibits differentiation and apoptosis of HL-60 cells: implications of RARα-mediated signals in myeloid leukemic cells

Retinoic acid (RA) induces HL-60 cells to differentiate terminally into mature granulocytes, which subsequently die by apoptosis. The biological effects of RA are mediated by two distinct families of transcription factors: retinoic acid receptors (RARs) and retinoid X receptors (RXRs). RARs and RXRs form heterodimers and regulate retinoid-mediated gene expression. We have recently developed a novel RAR-selective antagonist (ER27191) which prevents RAR activation by retinoids. Using this RAR-selective antagonist, and RXR and RAR agonist, we demonstrate the RAR-mediated signaling pathway is important for differentiation and apoptosis of myeloid leukemic cells. Simple activation of RXRs is not sufficient to induce apoptosis of the cells. Interestingly, the combination of the RAR-selective antagonist and 9-cis RA resulted in partial differentiation and apoptosis of HL-60 and NB4 cells, whereas the RAR antagonist completely blocked all-trans RA-induced differentiation and apoptosis of the cells. Additional experiments showed that levels of BCL-2 protein decreased during differentiation of myeloid leukemic cells. Furthermore, HL-60 cells transduced with a bcl-2 expression vector showed the same differentiation response to retinoids as did parental HL-60 cells even though apoptosis was inhibited in these bcl-2-transduced cells, suggesting that differentiation and apoptosis are regulated independently in myeloid leukemic cells.

Retinoid Resistance in Leukemic Cells

Recent studies have shown that a high proportion of patients with acute promyelocytic leukemia (APL) achieve complete remission after treatment with all-trans retinoic acid (RA). Nevertheless, despite an initial good response, most patients who received continuous treatment with all-trans RA relapsed and develop RA-resistant disease. The detailed mechanisms for this development of RA resistance by APL cells are still unclear. Several possible mechanisms have been considered to explain in vitro resistance to RA. One obvious explanation is the generation of new mutations in the retinoid receptors. However, UF-1 cells (the first permanent APL cell line with RA-resistant features) had no point mutations in the ligand-binding domain of the RAR-alpha gene. Another potential mechanism for clinical RA resistance is the pharmacologic alteration in the metabolism of all-trans RA. Continuous treatment with all-trans RA in APL is associated with a progressive reduction of the plasma concentrations of RA. Induction of cytochrome P-450, cellular RA-binding protein (CRABP) and P-glycoprotein resulted in lower plasma and cellular levels of active retinoids. Thus, acquired resistance to RA may be explained at least in part by drug metabolism in leukemic cells.

Novel variant isoform of G-CSF receptor involved in induction of proliferation of FDCP-2 cells: relevance to the pathogenesis of myelodysplastic syndrome.

Recent studies have shown that point mutations in granulocyte colony‐stimulating factor receptor (G‐CSFR) are involved in the pathogenesis of severe congenital neutropenia (SCN) and in the transformation of SCN to acute myelogenous leukemia (AML). It is reasonably speculated that the abnormalities in the signal transduction pathways for G‐CSF could be partly responsible for the pathogenesis and the development to AML in patients with myelodysplastic syndromes (MDS). Therefore, we investigated the structural and functional abnormalities of the G‐CSFR in 14 patients with MDS and 10 normal subjects. In in vitro colony forming assay, MDS samples showed reduced response to growth factors. However, G‐CSF, but not GM‐CSF and IL‐3, enhanced clonal growth in three cases of high risk patients with MDS (RAEB, RAEB‐t, and MDS having progressed to acute myeloid leukemia (AML)) and one low risk patient (RA). Eight out of 14 patients including above 4 patients demonstrated a common deletion of the G‐CSFR cDNA; a deletion of three nucleotides (2128–2130) in the juxtamembrane domain of the G‐CSFR, which resulted in a conversion of Asn630Arg631 to Lys630. To assess the functional activities of this deletion in the G‐CSFR isoform, a mutant with the same three‐nucleotide deletion was constructed by site‐directed mutagenesis. FDCP‐2 cells expressing the G‐CSFR isoform responded to G‐CSF, and exhibited proliferative responses than did those cells having wild‐type G‐CSFR. Moreover, these isoforms showed prolonged activation of STAT3 in response to G‐CSF than did the wild‐type. These results suggest that the deletion in the juxtamembrane domain of the G‐CSFR gives a growth advantage to abnormal MDS clones and may contribute to the pathogenesis of MDS.