Differential gene expression, GATA1 target genes, and the chemotherapy sensitivity of Down syndrome megakaryocytic leukemia.

Children with Down syndrome (DS) with acute megakaryocytic leukemia (AMkL) have very high survival rates compared with non-DS AMkL patients. Somatic mutations identified in the X-linked transcription factor gene, GATA1, in essentially all DS AMkL cases result in the synthesis of a shorter (40 kDa) protein (GATA1s) with altered transactivation activity and may lead to altered expression of GATA1 target genes. Using the Affymetrix U133A microarray chip, we identified 551 differentially expressed genes between DS and non-DS AMkL samples. Transcripts for the bone marrow stromal-cell antigen 2 (BST2) gene, encoding a transmembrane glycoprotein potentially involved in interactions between leukemia cells and bone marrow stromal cells, were 7.3-fold higher (validated by real-time polymerase chain reaction) in the non-DS compared with the DS group. Additional studies confirmed GATA1 protein binding and transactivation of the BST2 promoter; however, stimulation of BST2 promoter activity by GATA1s was substantially reduced compared with the full-length GATA1. CMK sublines, transfected with the BST2 cDNA and incubated with HS-5 bone marrow stromal cells, exhibited up to 1.7-fold reduced cytosine arabinoside (ara-C)-induced apoptosis, compared with mock-transfected cells. Our results demonstrate that genes that account for differences in survival between DS and non-DS AMkL cases may be identified by microarray analysis and that differential gene expression may reflect relative transactivation capacities of the GATA1s and full-length GATA1 proteins.

GATA1, cytidine deaminase, and the high cure rate of Down syndrome children with acute megakaryocytic leukemia.

Down syndrome children with acute megakaryocytic leukemia (AMkL) have higher cure rates than non-Down syndrome acute myeloid leukemia (AML) patients treated with cytosine arabinoside (ara-C). Megakaryoblasts from Down syndrome AML patients are more sensitive in vitro to ara-C than cells from non-Down syndrome AML patients. Somatic mutations in the GATA1 transcription factor have been detected exclusively and almost uniformly in Down syndrome AMkL patients, suggesting a potential linkage to the chemotherapy sensitivity of Down syndrome megakaryoblasts. Stable transfection of wild-type GATA1 cDNA into the Down syndrome AMkL cell line CMK resulted in decreased (8- to 17-fold) ara-C sensitivity and a threefold-lower generation of the active ara-C metabolite ara-CTP compared with that for mock-transfected CMK cells. High intracellular levels of uridine arabinoside (ara-U) (an inactive ara-C catabolite generated by cytidine deaminase) and cytidine deaminase transcripts were detected in GATA1-transfected CMK sublines, whereas no ara-U was detected in mock-transfected cells. Cytidine deaminase transcripts were a median 5.1-fold (P = .002) lower in Down syndrome megakaryoblasts (n = 16) than in blast cells from non-Down syndrome patients (n = 56). These results suggest that GATA1 transcriptionally upregulates cytidine deaminase and that the presence or absence of GATA1 mutations in AML blasts likely confers differences in ara-C sensitivities due to effects on cytidine deaminase gene expression, which, in turn, contributes to the high cure rate of Down syndrome AMkL patients.

CSP41a, a multifunctional RNA-binding protein, initiates mRNA turnover in tobacco chloroplasts.

Expression of chloroplast stem-loop binding protein (CSP)41a, a highly conserved chloroplast endoribonuclease, was reduced >90% by the expression of antisense RNA in Nicotiana tabacum. The most striking effects of this silencing were two- to sevenfold decreases in the degradation rates of rbcL, psbA, and petD transcripts in lysed chloroplast extracts. These results are consistent with the hypothesis that CSP41a participates in initiating mRNA turnover through endonucleolytic cleavages. Surprisingly, rbcL and psbA mRNAs accumulated to similar levels in wild-type and antisense lines. This suggested that decreased degradation was compensated by reduced transcription, which was confirmed using run-on transcription assays. The collective accumulation of petD-containing mRNAs in antisense plants decreased by 25% compared to wild-type controls. However, the relative levels of petD processing intermediates in wild-type and antisense plants did not differ, and there were no changes in petD 3'-end maturation, suggesting that CSP41a is not required for petD RNA processing. CSP41a is a Mg2+-dependent enzyme; therefore, extracts from antisense plants were tested at different Mg2+ concentrations. These experiments showed that the half-life of rbcL decreased as the Mg2+ concentration was reduced, and at <1 mm free Mg2+, conditions where CSP41a is nearly inactive in vitro, the rbcL degradation rate was similar in wild-type and antisense extracts, suggesting that CSP41a is normally bypassed under these conditions. Mg2+ has been shown to mediate RNA stability during chloroplast biogenesis, and our data suggest that regulation of CSP41a activity by Mg2+ is a component of this process.

Volatile isoprenoid constituents of fruits, vegetables and herbs cumulatively suppress the proliferation of murine B16 melanoma and human HL-60 leukemia cells.

Substantial evidence from epidemiological studies supports the inverse association between the intake of fruits, vegetables and other plant products and cancer incidence. Cancer-preventive constituents of fruits and vegetables may inhibit carcinogen activation, enhance carcinogen detoxification, prevent carcinogens from interacting with critical target sites, or impede tumor progression. These activities, however, are achievable only when levels of individual bioactive constituents reach beyond those attainable from a normal balanced diet. Isoprenoids, a broad class of mevalonate-derived phytochemicals ubiquitous in the plant kingdom, suppress the proliferation of tumor cells and the growth of implanted tumors. A search for volatile isoprenoid constituents of food products spanning seven plant families identified 179 isoprenoids. Of these, 41 purchased from commercial sources were screened for efficacy in suppressing the proliferation of murine B16 melanoma cells. Individual isoprenoids suppressed the proliferation of B16 and HL-60 promyelocytic leukemia cells with varying degrees of potency. Cell cycle arrest at the G(0)-G(1) phase and apoptosis account, at least in part, for the suppression. Blends of isoprenoids suppressed B16 and HL-60 cell proliferation with efficacies equal to the sum of the individual impacts. These findings suggest that the cancer-protective property of fruits, vegetables, and related products is partly conferred by the cumulative impact of volatile isoprenoid constituents.