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PubMed1999

Microsatellite instability and p53 mutations are associated with abnormal expression of the MSH2 gene in adult acute leukemia.

Microsatellite instability (MSI) and p53 mutations have been reported to occur in a significant proportion of patients with therapy-related acute myeloid leukemia (AML). MSH2 is one of the genes involved in DNA mismatch repair to maintain fidelity of genomic replication, and defects of MSH2 are directly involved in MSI in hereditary nonpolyposis colorectal tumors and other human tumors. We have examined the expression of MSH2 protein by Western blotting in 43 adult leukemia samples, including 42 AML and 1 acute lymphoblastic leukemia (ALL) using the antibody MSH2 (Ab-1) (Calbiochem, La Jolla, CA). Abnormal expression of MSH2 protein was found in 14 of 43 (32.6%) cases; a control antibody to actin was always positive. Of the 14 patients that had abnormal expression of MSH2, 2 had therapy-related acute leukemia and 9 were elderly patients (>60 years of age). Expression of MSH2 mRNA was further examined by reverse transcriptase-polymerase chain reaction (RT-PCR). Deletion of MSH2 mRNA was found in 1 of 14 cases with deficient MSH2 protein expression. This group of patients was also screened for loss of heterozygosity (LOH) at the MSH2 locus using a panel 4 microsatellite markers (D2S367, D2S288, D2S391, and D2S2294). LOH was found in 5 of 11 cases examined. There was no evidence of LOH in 14 patients with normal MSH2 expression who were examined using the same markers. Functional evidence for defective DNA mismatch repair in leukemic cells lacking MSH2 as manifest by MSI was found in 7 of 11 cases studied. Mutations of the p53 gene in these 43 samples were also investigated by direct sequencing of full-length p53 cDNA. Mutations of p53 were found in 6 of 43 cases, including 5 of the 14 (35.7%) cases that did not express MSH2 protein. In contrast, mutation of p53 was only found in 1 of 29 (3.4%) cases with normal MSH2 protein expression (chi2 = 5.720, P <.02). These results suggest that abnormalities of DNA mismatch repair due to defective MSH2 expression could play a key role in leukemogenesis, in particular in AML arising in elderly patients or secondary to previous chemotherapy.

Acute Disease↗
PubMed1996

Role of c-jun induction in the glucocorticoid-evoked apoptotic pathway in human leukemic lymphoblasts.

Accumulated evidence suggests tI AP-1 family in CEM cell clones exposed to the glucocorticoid dexamethasone (Dex) has been investigated. Dex is known to cause apoptosis of lymphoid cells in general and of sensitive human lymphoid CEM cell clones in particular. This study finds that Dex induces c-jun mRNA and cJun protein in cells of the sensitive clone CEM-C7 and of the lysis-sensitive OEM hybrid clone H10. CEM-O7 cells were screened for several other Jun/Fos family proteins. Both cFos and JunD were expressed but were unaffected by the steroid, and JunB was not detected. In the continual presence of Dex the induction of cJun began about 6 h after addition of Dex, reached a maximum by 24 h, and plateaued for 72 h, while cell death did not begin until 24-48 h. In clone OEM-Cl cells, which contain glucocorticoid receptor (GR) but are resistant to lysis by Dex, the basal, and even the fully induced, cJun levels are below the basal levels in OEM-07 and H10 cells. To test the hypothesis that cJun plays an important role in steroid-evoked apoptosis, stable transfectants expressing Dex-regulable antisense c-jun RNA were established. Mass cultures of these cells showed reduced sensitivity to Dex, and in three of three clones tested, complete resistance to Dex was obtained. This occurred even though endogenous genes (GR, c-jun) normally responsive to Dex were still inducible, indicating that the GR and basic glucocorticoid response apparatus were intact. It is concluded that Dex induces cJun levels in sensitive OEM cells before cell death and that this induction plays a role in the apoptotic process.

Apoptosis↗
PubMed2006

Retroviral integration site analysis identifies ICSBP as a collaborating tumor suppressor gene in NUP98-TOP1-induced leukemia.

INTRODUCTION: The NUP98-TOP1 fusion gene is one of 18 distinct translocations identified in acute myeloid leukemia involving the N-terminal portion of the nucleoporin NUP98. We previously reported that expression of NUP98-TOP in murine bone marrow induces a lethal, transplantable leukemia. However, the long latency suggests the in vivo acquisition of additional mutations and/or time required for clonal outgrowth of rare transformed cells arising from the collaboration of NUP98-TOP1 and a cooperating event. The aim of this study was to test whether retroviral insertional mutagenesis contributes to disease onset and whether integration site analysis can identify collaborating genes. METHODS: The genomic sites of retroviral integration in NUP98-TOP1-induced leukemic mice were analyzed. This screen identified a proviral integration that disrupts expression of the Interferon consensus sequence binding protein (ICSBP) tumor suppressor gene. Intriguingly, an ICSBP deficiency induces a chronic myeloid leukemia-like disease in mice and its reduced expression has been observed in several human leukemias. To ascertain whether an ISCBP deficiency collaborates with NUP98-TOP1 in leukemogenesis, we expressed NUP98-TOP1 in ICSBP(-/-) bone marrow. RESULTS: The in vivo myeloproliferation induced by NUP98-TOP1 was markedly exaggerated with the ICSBP(-/-) deficiency. Moreover, NUP98-TOP1/ICSBP(-/-) mice had a reduced survival compared with NUP98-TOP1/ICSBP(+/+) mice. CONCLUSION: These results reveal the novel finding of collaboration between the ICSBP tumor suppressor gene and NUP98-TOP1 in leukemogenesis. Moreover they further illustrate the power of retroviral integration site analysis for identifying novel cooperating tumor suppressor genes.

Animals↗
PubMed1999

Leukemia patient-derived lymphoblastoid cell lines exhibit increased induction of leukemia-associated transcripts following high-dose irradiation.

Improvement in diagnostic cytogenetic techniques has led to the recognition of an increasing number of leukemia-associated chromosomal translocations and inversions. These genetic lesions frequently are associated with the disruption of putative transcription factors and the production of hybrid transcripts that are implicated in leukemogenesis. Epidemiologic evidence suggests that some, but not all, individuals with a history of gamma-irradiation exposure are at increased risk of developing chronic myeloid leukemia (CML). CML is characterized by the Philadelphia chromosome and transcription of the resulting hybrid BCR-ABL gene. Utilizing the leukemia-associated BCR-ABL p210 transcript as a marker, we sought differences in the induction of illegitimate genetic recombination following high-dose gamma-irradiation of karyotypically normal lymphoblastoid cell lines (LCL) derived from individuals with and without a history of myeloid leukemias. Six LCL [4 leukemia patient derived [2 acute myeloid leukemia and 2 CML] and 2 from normal individuals were analyzed with reverse transcriptase polymerase chain reaction for BCR-ABL under stringent conditions following exposure to 0, 50, or 100 Gy of LET gamma-irradiation delivered via a Varian linear accelerator at 4 MV. Transcripts identical to disease-associated b2a2 and b3a2 transcripts were detected both spontaneously (background illegitimate genetic recombination) and following gamma-irradiation. Background BCR-ABL positivity was demonstrable in 4 of the 6 LCL, with no significant difference in detection between leukemic- and nonleukemic-derived LCL. Overall, increasing gamma-irradiation dose resulted in an increased frequency of BCR-ABL transcript detection (0 Gy vs 50 Gy vs 100 Gy,p = 0.0023, Chi-square test). Within the leukemic- but not the nonleukemic-derived LCL there was significantly greater BCR-ABL positivity after gamma-irradiation compared to unirradiated equivalents. Furthermore, the BCR-ABL positivity of both the AML- and CML-derived LCL after gamma-irradiation was significantly greater than that of the nonleukemic-derived LCL after gamma-irradiation. We speculate that this difference in the detection of illegitimate after gamma-irradiation recombination may be due to aberrant DNA double strand break repair mechanisms in individuals predisposed to the development of myeloid leukemias.

Acute Disease↗
PubMed1994

Pattern of expression of five alternative transcripts of the lck gene in different hematopoietic malignancies: correlation of the level of lck messenger RNA I B with the immature phenotype of the malignancy.

The lck gene encodes a tyrosine protein kinase of the src family which is highly expressed in T-lymphocytes. Two widely separated promoters govern expression of the lck gene. We report in this study that alternative splicing between cryptic donor and acceptor sites in the 5' untranslated region of the transcripts initiated at the type I promoter give rise to three different type I lck mRNAs, I A, I B, and I C. Altogether with the types II A and II B reported previously, the lck transcription is thus characterized by five alternative transcripts. We further used the complementary DNA-polymerase chain reaction assay to describe the pattern of expression of these five lck transcripts in different hematopoietic cell lines and in blood or bone marrow samples from healthy donors and leukemic patients. We report that the transcript II A is by far the major transcript present both in human samples and in T-cell lines. The low lck expression in B-cell lines is characterized by the quite exclusive presence of the transcript I B. We show that hematopoietic diseases characterized by the presence of immature cells [acute myeloid leukemia (AML-0 and AML-1) and T- and B-cell acute lymphoid leukemia] exhibit a marked increase of the transcript I B. No significant difference from the normal pattern is observed in AML according to the differentiation stage (AML-2 to AML-5). A normal pattern of lck expression is restored in AML-0 and AML-1 patients at complete remission.

Adolescent↗
PubMed1990

Expression of the mdr3 gene in prolymphocytic leukemia: association with cyclosporin-A-induced increase in drug accumulation.

Typical multidrug resistance in human and animal cell lines is caused by overactivity of an unidirectional transmembrane drug efflux pump, encoded by the MDR genes, called mdr genes in mice and humans and pgp genes in hamsters. In humans, two mdr genes, mdr1 and mdr3, with approximately 80% nucleotide homology, have been identified. There is increasing evidence that overexpression of the mdr1 gene plays a role in resistance to anticancer agents in specific tumor types. However, currently no data are available on a possible role for mdr3 in drug resistance. Here we report high levels of expression of mdr3 gene sequences in leukemic cells from 6 out of 6 patients with prolymphocytic leukemia (PLL). No mdr1 expression was detected in 5 out of 6 of these samples, whereas a low level of mdr1 expression was found in a sample from one PLL patient in the course of transformation to non-Hodgkin's lymphoma. Except for this patient, all other PLL cases studied had not received prior chemotherapy. In vitro drug uptake studies showed that daunorubicin accumulation in PLL cells was increased by cyclosporin A. Since cyclosporin A is an inhibitor of the mdr1-encoded P-glycoprotein drug pump, these data suggest that in PLL cells mdr3 also codes for a drug efflux pump. Our findings could partly explain the primary refractoriness of PLL to chemotherapy.

Adult↗
PubMedDate not supplied

Activation of expression of p15, p73 and E-cadherin in leukemic cells by different concentrations of 5-aza-2'-deoxycytidine (Decitabine).

BACKGROUND: Inactivation of genes that suppress neoplasia by aberrant DNA methylation is a key event that occurs during the development of leukemia. The inhibitor of DNA methylation, 5-aza-2'-deoxycytidine (5AZA), which can re-activate these genes, is under clinical investigation for therapy of leukemia. The objective of this study was to determine the concentrations of 5AZA that will re-activate target silent genes in human leukemic cell lines. MATERIALS AND METHODS: RT-PCR was used to evaluate the effect of concentrations of 1 to 100 ng/ml of 5AZA on the re-activation of p15 and p73 in KG1a myeloid leukemic cells and E-cadherin in HL-60 myeloid leukemic cells. The effect of 5AZA on inhibition of growth, DNA synthesis and colony formation in these cell lines was also investigated. RESULTS: The extent of activation of the target genes was dependent on the concentration of 5AZA. For p15, pronounced activation was observed at 10 ng/ml or greater. For p73 and E-cadherin significant activation was observed at 100 ng/ml of 5AZA. Maximal inhibition of growth, DNA synthesis and colony formation occurred at 100 ng/ml. CONCLUSION: The in vitro antineoplastic and gene re-activation activity of 5AZA is dependent on the concentration of this analog. These data may be helpful in the design of the optimal dose-schedule of 5AZA for the clinical therapy of leukemia.

Antimetabolites, Antineoplastic↗
PubMedDate not supplied

Relationship between MDR1 gene and surface markers in acute myeloid leukemia.

The MDR1 gene is of prognostic significance in acute myeloid leukemia (AML). The relationship of this gene to surface markers largely remains unclear. Therefore, we have studied the association of MDR1 gene expression with the expression of specific surface markers in AML. MDR1 RNA expression of leukemic cells was determined by slot blot analysis. Expression of P-glycoprotein and surface markers (CD7, CD13, CD19, CD34, HLA-DR, TdT, blood group H) was assessed by immunocytochemistry. MDR1 RNA (n = 79) and P-glycoprotein (n = 52) expression were detected in 63% and 63% of the patients, respectively. CD7, CD13, CD19, CD34, HLA-DR, TdT and blood group H were positive in 17%, 84%, 0%, 51%, 82%, 11% and 11% of the patients. MDR1 RNA or P-glycoprotein expression were not associated with the expression of either CD7, CD13, CD19, CD34, TdT or blood group H. However, P-glycoprotein expression was more frequent in HLA-DR positive than in HLA-DR negative patients (47% versus 10%, p = 0,04). Consistent with the latter finding, patients with intermediate or high MDR1 RNA expression expressed HLA-DR more frequently than patients with negative or weak MDR1 RNA expression (96% versus 76%, p = 0,03). In conclusion, MDR1 gene expression of AML cells was independent of surface markers except HLA-DR.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1992

Genomic organization of the putative human homeobox proto-oncogene HOX-11 (TCL-3) and its endogenous expression in T cells.

The HOX-11 (TCL-3) gene, which is abnormally expressed in the leukemic cells of some patients with T-cell acute lymphoblastic leukemia, is a new member of the homeobox gene family. It is structurally altered by the t(10;14) chromosomal translocation, resulting in head-to-tail juxtaposition of HOX-11 with the T-cell receptor delta-chain gene. In order to understand the normal functions of HOX-11 and its role in T-cell leukemia, we have determined the exon-intron structure of the HOX-11 gene. By using oligonucleotide primers flanking an intron of the HOX-11 gene, we have developed a quantitative reverse transcription-polymerase chain reaction (RT-PCR) assay for the expression of HOX-11. We detected HOX-11 expression in multiple cell lineages including normal T cells and two T-cell lines in which the HOX-11 gene appeared to be unaltered in structure. Our results suggest that deregulation of the endogenous expression of HOX-11 in normal T cells represents an essential step towards the formation of this type of T-cell leukemia.

Amino Acid Sequence↗
PubMed2001

Expression of tumor-suppressor genes interferon regulatory factor 1 and death-associated protein kinase in primitive acute myelogenous leukemia cells.

Previous studies indicate that human acute myelogenous leukemia (AML) arises from a rare population of leukemic stem cells. Cells of this nature can initiate and maintain leukemic cell growth in both long-term cultures and nonobese diabetic/severe combined immune-deficient mice. To characterize the biology of primitive AML cells, gene expression screens were performed with 7 primary AML and 3 normal specimens. For each sample, stem cell populations (CD34(+)/CD38(-)) were isolated and used to synthesize radiolabeled complementary DNA (cDNA). AML vs normal probes were then hybridized to cDNA arrays containing genes related to cancer and apoptosis. Of approximately 1400 genes analyzed, 2 tumor-suppressor genes were identified that were overexpressed in all 7 of the AML CD34(+)/CD38(-) cell populations: death-associated protein kinase and interferon regulatory factor 1. Expression of each gene was confirmed by reverse-transcription polymerase chain reaction and immunoblot analysis. It is proposed that tumor-suppressor proteins play a role in the biology of primitive AML cells. (Blood. 2001;97:2177-2179)

ADP-ribosyl Cyclase↗
PubMed1989

Analysis of molecular events in leukemic cells arrested at an early stage of T-cell differentiation.

We analyzed the rearrangement and expression of T-cell receptor (TCR) genes, including the recently identified TCR delta gene, in 21 patients with T-lineage leukemia/lymphoma. Among 8 patients with CD3-, CD4-, and CD8- (group I), 2 patients showed germline configuration of the TCR delta, gamma, beta, and alpha genes and 1 patient demonstrated only TCR delta gene rearrangement. All nine patients with CD3-, CD4+, and/or CD8+ (group II) showed concomitant rearrangements of the TCR delta, gamma, and beta genes. TCR alpha gene rearrangement was also observed in two patients. Three of four patients with CD3+ (group III) showed rearrangement of the TCR alpha gene with deletion of both alleles or of a single allele of the TCR delta gene. With Northern blot analysis, full-length transcripts of the TCR delta gene were detected in 3 of 15 examined patients. All were restricted to group I or group II. In contrast, full-length transcripts of TCR beta and alpha were observed mainly in samples from groups II and III. Based on these findings, rearrangement of the TCR delta gene may be the earliest event in T-cell differentiation, preceding rearrangements of the other TCR genes.

Antigens, CD↗
PubMed2003

E mu-BRD2 transgenic mice develop B-cell lymphoma and leukemia.

Transgenic mice with lymphoid-restricted overexpression of the double bromodomain protein bromodomain-containing 2 (Brd2) develop splenic B-cell lymphoma and, upon transplantation, B-cell leukemia with leukemic infiltrates in liver and lung. Brd2 is a nuclear-localized transcription factor kinase that is most closely related to TATA box binding protein-associated factor, 250 kDa (TAF(II)250) and the Drosophila developmental protein female sterile homeotic. Constitutive expression of BRD2 in the lymphoid compartment increases cyclin A transcription, "priming" transgenic B cells for proliferation. Mice stochastically develop an aggressive B-cell lymphoma with the features of B-1 cells, including CD5 and surface IgM expression. The B-cell lymphoma is monoclonal for immunoglobulin gene rearrangement and is phenotypically stable. The lymphoblasts are very large and express a transcriptome that is similar to human non-Hodgkin lymphomas. Both a wild-type BRD2 transgene and a kinase-null point mutant drive lymphomagenesis; therefore we propose that, rather than kinase activity, Brd2-mediated recruitment of E2 promoter binding factors (E2Fs) and a specific histone acetyltransferase to the cyclin A promoter by both types of transgene is a mechanistic basis for neoplasia. This report is the first to describe a transgenic mouse model for constitutive expression of a protein with more than one bromodomain.

Animals↗
PubMedDate not supplied

Correlation of minimal residual disease by assessing Wilms tumor gene expression and engraftment by variable number of tandem repeats in children with leukemia posthematopoietic stem cell transplantation.

An important measure to ensure successful follow-up in patients with allogeneic stem cell transplant is to evaluate for engraftment. Recent studies have shown that detecting minimal residual disease is important in order to predict early clinical relapse. We followed 88 leukemic patients with pre- and posttransplant Wilms tumor gene (WT1) levels to predict relapse and variable number of tandem repeats (VNTR) for engraftment. We have found that high pretransplant WT1 levels correlated significantly with relapse in all patient groups, but more significantly in the acute nonlymphoblastic leukemia (ANLL) patients. Posttransplant WT1 level correlated with VNTR status such that low WT1 is associated invariably with VNTR of 100% donor origin, while high WT1 is associated with VNTR of 20%. The association is significant in all patients, specifically in ANLL patients. In this preliminary study, we demonstrate that patients harboring detectable levels of WT1 prior to stem cell transplant have a higher chance of relapse, and posttransplant WT1 and VNTR status appeared to be dependent parameters predicting relapse when present in the posttransplant period. By combining 2 highly sensitive molecular techniques, we have found that this combined technique provided us with a promising alternative for overcoming the limitations imposed by each separate procedure. More studies are necessary before we can come to any significant conclusions.

Adolescent↗
PubMed1996

Interleukin-10 gene expression in adult T-cell leukemia.

We studied the serum levels of interleukin-10 (IL-10), in patients with adult T-cell leukemia (ATL) caused by human T-cell leukemia virus type I (HTLV-I) infection. Elevated IL-10 levels were observed in 33 of 45 patients with ATL. Fresh leukemic cells from ATL patients as well as HTLV-I-infected T-cell lines MT-2, SLB-1, and C10/MJ expressed IL-10 mRNA by reverse transcription-polymerase chain reaction analysis, whereas IL-10 mRNA was not detected in normal peripheral mononuclear cells and an uninfected T-cell line Jurkat. IL-10 protein was also detected in the culture medium of leukemic cells from ATL patients as well as these HTLV-I-infected cell lines, and in the extracellular fluids of ATL patients. Interestingly, MT-4 cells, which did not express Tax although transformed by HTLV-I, did not express IL-10 at either the mRNA or protein level. To elucidate the role of the HTLV-I encoded transactivator Tax in IL-10 gene expression, Jurkat cells were transfected with a Tax expression plasmid. In transiently transfected Jurkat cells, endogenous IL-10 mRNA expression was induced by Tax. Stably transfected Jurkat cell lines expressed IL-10 mRNA and secreted IL-10 protein into the culture medium. The nuclear factor (NF)-kappa B pathway is a target for Tax transactivation. We treated MT-2 cells with phosphorothioate antisense oligonucleotides to the p65 subunit of NF-kappa B. A reduction in the expression of p65 was accompanied by a reduction in IL-10 gene expression and IL-10 production. We showed that the IL-10 kappa B-like sites ( kappa B1,-2,034 to -2,025; kappa B2, -1,961 to -1,952; kappa B3, -452 to -443) specifically formed a complex with NF-kappa B-containing nuclear extract from MT-2 cells and that NF-kappa B bound with the highest affinity to the kappa B2 element (kappa B2 > kappa B3 > kappa B1). These data suggest a general role for NF-kappa B activation in the induction of IL-10 gene transcription. Activation of IL-10 in HTLV-I-infected cells may contribute to the pathology associated with HTLV-I infection.

Adult↗
PubMed1994

Interferon-alpha-2b downregulation of oncogenes H-ras, c-raf-2, c-kit, c-myc, c-myb and c-fos in ESKOL, a hairy cell leukemic line, results in temporal perturbation of signal transduction cascade.

ESKOL, a B-lymphoblastoid cell line consisting of late differentiated cells, resembles hairy cell leukemia (HCL). It is pseudodiploid with a deleted 7q and an unbalanced translocation between chromosomes 4 and 6. It was screened by Northern hybridization for oncogenes, including H-ras, c-raf-2 (c-raf1p1), c-kit, c-myc, c-myb, c-fos, Fim-1, c-jun, ski, and c-mos, which are believed to contribute to B-cell differentiation and maturation. Interferon-alpha-2b (IFN) downregulates the expression of H-ras, c-raf-2, c-kit, c-myc, c-myb, c-fos, as determined by Northern hybridization of RNA isolated from cells harvested at time points during a 30 h time course. Downregulation of oncogenes H-ras, c-raf-2, c-kit, whose proteins are associated with cell surfaces or are cytosolar transducers, occurs before those oncogenes c-myc, c-myb, and c-fos, whose products are DNA binding proteins. This suggests a temporal perturbation of signal transduction by IFN. No change in oncogene expression occurred in non-treated cells nor were these oncogenes expressed in the non-transformed B-lymphoblast cell line, Wil-2, under the same treatment regimen. The basis for the IFN perturbation is not understood; yet the role of these oncogenes as signal transducers in differentiation and proliferation of human hematopoietic progenitors is unfolding, and ESKOL is an excellent system in which to study this phenomenon.

Aged↗
PubMed1998

Ratio of bcl-xshort to bcl-xlong is different in good- and poor-prognosis subsets of acute myeloid leukemia.

BACKGROUND: Acute myeloid leukemia (AML) is a heterogeneous collection of leukemic disorders ranging from chemotherapy-sensitive subsets [inversion 16 and t(8;21)], which often can be cured with cytosine arabinoside alone, to the most resistant subsets, which can survive even supralethal levels of combination alkylator chemotherapy (cytogenetic subsets monosomy 5 and monosomy 7). MATERIALS AND METHODS: To analyze the expression of BCL-2 family genes, which are expressed in these subsets of AML, we used PCR sequence amplification reactions that are dependent on oligonucleotide primers representing the BH1 and BH2 homology domains to generate the unique regions between BH1 and BH2. These primers are conserved among all members of the BCL-2 gene family and are separated by a 150 nucleotide region sequence between the BH1 and BH2 domains. The PCR products unique to each BCL-2 family member were cloned directionally into sequencing vectors. The identity of the insert of each clone was determined by slotblots of the DNA amplified from individual colonies and by hybridization with radioactive probes specific to the bcl-2, bcl-x, or bax genes. RESULTS: We found that bcl-2 is the predominant member expressed in AML samples with a poor prognosis (-5, -7), whereas the transcripts of bcl-x are higher than those of bcl-2 in the AML samples with a good prognosis [inv16, t(8;21)]. No significant difference in bax expression was detected between AML subsets of good and bad prognosis. The ratio of bcl-xlong, which inhibits apoptosis, to bcl-xshort, which promotes apoptosis, was determined by amplification with a pair of primers specific to bcl-x followed by separation of the PCR product on agarose gels. Bcl-xlong and bcl-xshort appeared as bands of different molecular mass on a molecular weight gel and were visualized by ethidium bromide staining or Southern blot analysis with a bcl-x-specific probe. CONCLUSIONS: We found that the ratio of bcl-x long to bcl-x short was higher in the AML patients with a poor prognosis. These experiments showed that the levels of BCL-2 family members in the leukemia cells of good- and poor-prognosis subsets are different. In addition, novel members of the BCL-2 family were isolated from the cells of AML patients of either prognosis.

Acute Disease↗
PubMed1996

Expression of a cosmid containing the LCR, (A)gamma, delta, and beta globin genes in mouse erythroleukemia cells.

A novel method involving the packaging of phage particles was used to introduce cosmids containing components of the human beta-globin locus control region (LCR) and the human (A)gamma, delta, and beta globin genes into mouse erythroleukemia (MEL) cells. After stable transfection, MEL clones were selected and analyzed for expression of human genes. Both (A)gamma and beta globin mRNA were expressed in these clones, indicating that MEL cells do not suppress transcription of the human gamma globin gene. The variability of human (A)gamma vs. beta globin expression from clone to clone prevents a clear delineation of differences in the expression of these two genes with a cosmid in which a region potentially involved in gamma-to-beta switching has been deleted. The results suggest that MEL cells are capable of supporting human gamma expression, despite their predominantly adult phenotype.

Adult↗
PubMed2000

Cellular expression of MDM2 and p53 in childhood leukemias with poor prognosis.

BACKGROUND: Previous studies have suggested that altered expression or dysfunction of the tumor suppressor gene p53 or the oncogene MDM2 could indicate disease progression in children with leukemia who would fail to achieve complete remission or who would relapse. While these studies mainly have described aberrations of MDM2 and p53 function at the DNA and mRNA-level, we have examined p53 and MDM2 expression at the protein level. Mutation of the p53 tumor suppressor gene may result in cellular accumulation of the p53 protein, due to prolonged half-life of the abnormal protein. The p53 protein can also be rendered nonfunctional by overexpression of proteins that bind to p53, such as MDM2. Both pathways have been proposed to disrupt cell cycle regulation in humans. Recent studies have shown that increased expressions of MDM2 as well as of p53 can be detected at the protein level in the absence of gene amplification. PROCEDURE: Forty-three bone marrow samples were analyzed immunohistochemically for p53 and MDM2. Twenty-nine bone marrow samples were obtained in children with active, prognostically unfavorable leukemia and MDS. Fourteen bone marrow samples were from children with non-malignant hematological disorders. RESULTS: p53 protein was expressed in 12 patients and MDM2 in 17 patients with leukemia. In the control group MDM2 expression was detected in one child, while p53 was not found in any of the samples. CONCLUSIONS: Our findings of p53 or MDM2 positive cells in a majority of children with unfavorable prognostic features suggests that dysfunction of the p53-dependent cell growth control have a role in the development of high risk leukemias.

Adolescent↗