Biomedical subjects

M D Minden

Publications and source records attributed to M D Minden.

At least 55 records · Page 3Linked to original sources

PubMed1996

Post-transcriptional regulation of bcl-2 in acute myeloblastic leukemia: significance for response to chemotherapy.

The blast stem cells of acute myeloblastic leukemia become more sensitive in culture to the chemotherapeutic agents cytosine arabinoside (Ara-C) and daunorubicin (DNR) when exposed to all-trans retinoic acid (ATRA) after drug. We have proposed that down regulation of bcl-2 by ATRA is part of the mechanism of sensitization. The hypothesis is based on reduced expression of bcl-2 mRNA, as seen in Northern blots, after ATRA. Nuclear run on experiments, however, failed to account completely for the effect at the transcriptional level. Accordingly, we looked for post-transcriptional effects of ATRA on bcl-2, using metabolic labelling of the protein to measure stability. We found that the half-life of bcl-2 protein is markedly shortened after treatment with ATRA. Hydrocortisone (HC) protects cells against the toxic effects of Ara-C or DNR when given before drug. HC does not alter bcl-2 expression at the level of mRNA; however, metabolic labelling shows that newly synthesized bcl-2 protein is stabilized in blast cells treated with HC. Response to Ara-C by growth factor responsive blast cells is influenced by the factor in the cultures; cells are more sensitive in cultures with G-CSF and less sensitive when GM-CSF is present. We compared two blast cell lines, OCI/AML-5, primarily responsive to GM-CSF, and OCI/AML-10, primarily responsive to G-CSF. Growth factor did not influence the stability of bcl-2 protein in either line. In contrast, Western blots showed that the amount of bcl-2 protein was greater in cultures with GM-CSF or GM-CSF in combination with G-CSF than in cultures with G-CSF or no added factor. This pattern was seen regardless of the mitogenic response to G-CSF or GM-CSF. We interpret our findings as indicating that bcl-2 protein is transcriptionally activated; that the stability of the protein is decreased after ATRA and increased after HC; that the amount of bcl-2 protein is greater in cultures with GM-CSF than in cultures with G-CSF, regardless of which factor gives the greater mitogenic response. We propose that these post-transcriptional modifications of transcriptionally activated bcl-2 account, in part, for the regulation of drug sensitivity by ATRA, HC and growth factors.

Anti-Inflammatory Agents↗

PubMed1996

Response of the blast stem cells of acute myeloblastic leukemia to G-CSF, GM-CSF, or the ligand for C-KIT, alone or in combination.

Growth factors are commonly included in protocols for the treatment of acute myeloblastic leukemia (AML). Because the response of blast stem cells in culture to growth factors might influence the contribution of factor to clinical outcome, we studied 42 patients with AML or severe myelodysplasia. Peripheral blood blast cells were cultured in a clonogenic assay at three cell concentrations and with the following combinations of growth factors: no added growth factor (NF), G-CSF, GM-CSF, Kit ligand (KL), G-CSF + KL, GM-CSF + KL, and G-CSF + GM-CSF + KL. The slope of the line relating cell number plated to colony formation was calculated by least squares. The slopes were used to form three equally sized groups of patients. Marked heterogeneity was found in response of the blast populations to factor. A few general conclusions emerged: (1) autonomous blast populations are very rare; (2) although usually a population responds better to one of the growth factors than to others, seldom is the response exclusively to one factor; (3) when more than one factor is included in the cultures, synergism is usually seen. Significant associations were seen between successful remission induction for low slope values in cultures with NF or KL alone. For remission, but not survival, associations were found with intermediate values of slope in cultures with G-CSF + KL and GM-CSF + KL. We conclude that measurements of growth factor response are feasible and yield clinically useful data.

Adult↗

PubMed1996

Regulation of the synthesis of bcl-2 protein by growth factors.

The sensitivity of AML blast stem cells can be measured in cell culture, using a clonogenic assay to determine survival after each of a graded series of drug concentrations. For cytosine arabinoside, the dose-response curve is a simple negative exponential that can be described by a D10 value, a measure of slope. This D10 value can be affected by regulatory molecules added to the cultures. All-trans retinoic acid (ATRA) usually sensitizes cells, while hydrocortisone (HC) is protective. Growth factor responsive cells are more Ara-C sensitive in G-CSF than in GM-CSF or IL-3. The proto-oncogene bcl-2 may be part of the mechanism by which drug sensitivity is regulated. Previous work has shown that ATRA decreases bcl-2 RNA expression and the half-life of the protein; in contrast, the protein from cells treated with HC is more stable than controls. Growth factors were not shown to change either expression of bcl-2 RNA or the stability of its protein. In this paper, we describe experiments where OCI/AML-1 cells were grown in G-CSF and then transferred to medium containing both G-CSF and the GM-CSF-IL-3 fusion protein pIXY. Steady-state levels of bcl-2 protein were measured by Western blot and synthesis by incorporation of 35S methionine into protein. We observed that both measures doubled within 12-24 h after transfer from G-CSF in G-CSF with pIXY, but promptly returned to the previous state when pIXY was withdrawn. We conclude that growth factors regulate that activity of bcl-2 post-transcriptionally by altering the rate of synthesis of the protein.

Antimetabolites, Antineoplastic↗

PubMed1995

c-KIT expression enhances the leukemogenic potential of 32D cells.

The growth of human leukemic cells in culture and in vivo is dependent upon the presence of hematopoietic growth factors. Most populations of human leukemic acute myeloblastic leukemia (AML) cells express c-Kit on their surface and respond to Kit ligand (KL) in culture. To determine if this interaction was of potential significance in vivo we used a mouse model system. 32D cells, a murine IL-3-dependent myeloid cell line, were rendered KL responsive by transfection of the murine c-Kit. After injection of 32D or 32D-Kit cells into syngeneic hosts, animals bearing 32D-Kit cells, but not 32D cells, became moribund and were killed. These animals had circulating leukemic blast cells, infiltration of bone marrow, spleen, brain, liver, lung, and kidney. Cells recovered from some of the animals continued to be dependent upon IL-3 or KL for growth while in other cases the cells were factor independent. This model illustrates that the constitutive expression of c-Kit enhances the leukemic potential of 32D cells. The model will be useful for studying the progression of leukemia in vivo and testing whether interruption of the interaction of Kit and KL can affect the growth of leukemic cells.

Amino Acid Sequence↗

PubMed1995

p53 mutations, c-myc and bcl-2 rearrangements in human non-Hodgkin's lymphoma cell lines.

Fourteen Non-Hodgkin's lymphoma cell lines were generated and assessed for the presence of structural p53, c-myc and bcl-2 gene changes. Single or multiple changes were observed in 11 of the lines. Alterations of the p53 gene were most frequent and documented for 10 lines by immunoprecipitation using the antibodies PAb 240 and PAb 1801, sequencing studies and Southern blot analysis. A detailed study was performed in one of the cell lines (OCI-Ly 4) for which material of the original tumor sample was available. Two point mutations identified by sequencing cDNA derived from the cell line were also present in the original tumor specimen. In contrast, DNA prepared from fibroblasts of the same patient did not show the mutations. Six of the 14 lines demonstrated c-myc rearrangements, while bcl-2 changes were observed in 4. The presence of c-myc was associated with shorter survival of this group of patients with aggressive disease. None of the other changes present as single or composite alterations were correlated with clinical outcome measures.

Adult↗

PubMed1995

Molecular characterization of a chromosome translocation breakpoint t(11;14)(p13;q11) from the cell line KOPT-K1.

Recurrent chromosome translocations involving 11p13 and 14q11 are found in 5-10% of cases of T-ALL. The gene involved in the translocation on chromosome 14 is the T cell antigen receptor alpha or delta. The putative oncogene on chromosome 11 is rhombotin 2 (RBTN2)/translocated in T cell gene 2 (ttg-2), a member of the LIM family of proteins. In this paper we characterize a cell line KOPT-K1 that has a t(11;14)(p13;q11). The breakpoint on chromosome 11 involves an Alu-rich region with the break occurring between two Alu sequences on chromosome 11. In addition, approximately 70 bases from the break on chromosome 11 is a tetranucleotide repeat. Whether either of these structures played a role in the translocation is not known. No heptamer or nonamer sequences, implicated in other rearrangements were found near the breakpoint. The breakpoint on chromosome 11 maps more centromeric than previous translocations of this region. Despite this the RBTN2 gene is highly expressed in KOPT-K1. This cell line will be useful for investigating the role of RBTN2 in leukemogenesis and the mechanism by which the translocation alters the expression of RBTN2.

Base Sequence↗

PubMed1995

Sensitivities of AML blast stem cells to idarubicin and daunorubicin: a comparison with normal hematopoietic progenitors.

The objective of this study was to determine whether or not cell culture studies could contribute to the correct choice between idarubicin (IDA) and daunorubicin (DNR) for combination with ara-C in remission induction therapy of AML. Two growth factor-sensitive AML cell lines and the peripheral blood blast cells from 10 patients with AML were studied in culture for sensitivity to IDA and DNR under four culture conditions; cells were grown either in methylcellulose or suspension culture in the presence of G-CSF or GM-CSF. Normal bone marrow cells were cultured in methylcellulose in the presence of IDA or DNR under conditions suitable for the detection of BFU-E and CFU-C. Dose-response curves for AML blast cells were characterized by an initial shoulder and then an exponential decrease in survival. Marked patient-to-patient variation was observed for both portions of the survival curves. IDA was significantly more toxic to blast cells than DNR, especially for more sensitive cell populations. Consistent differences in drug sensitivity in the four culture conditions were not observed. BFU-E and CFU-C dose-response curves of normal marrow progenitors resembled those of AML blast cells but in contrast fell within a narrow range. The culture studies support the clinical finding in AML of modest superiority of IDA over DNR. The heterogeneity in sensitivity of AML blasts in culture suggests an opportunity to individualize treatment. Preclinical studies may help in developing such a therapeutic approach.

Aged↗

PubMed1995

Direct evidence for the participation of bcl-2 in the regulation by retinoic acid of the Ara-C sensitivity of leukemic stem cells.

All-trans retinoic acid (ATRA) increases the sensitivity of AML blast cells to cytosine arabinoside (Ara-C) or daunorubicin (DNR) when ATRA is given after drug. We have proposed that down-regulation of bcl-2 is part of the mechanism by which ATRA regulates drug sensitivity. To test this hypothesis cDNA encoding bcl-2 was transfected into cells of the continuous lines OCI/AML-2 and OCI/AML-5. Four transfectant lines were isolated; three contained transfected bcl-2 in the sense orientation (AML5-BCL2sa, AML5-BCL2sb and 2-bcl2) and one with anti-sense bcl-2(AML5-bcl2as). The presence of the transfected gene was demonstrated by Northern blot; translation of the sense transfected genes into protein was demonstrated by Western blotting. Lines with sense-oriented transfected bcl-2 were significantly less sensitive to Ara-C or H2O2 than the parental lines; the cells with anti-sense transfected genes were more sensitive than their parent but the difference did not reach statistical significance. The effect of ATRA on bcl-2 expression was compared in sense-transfected cells and their parents; by Northern blotting it was shown that the endogenous but not the transfected genes were down-regulated after ATRA exposure. The capacity of cells with transfected genes to respond to ATRA was tested by obtaining Ara-C survival curves for ATRA-treated cells. Compared to controls not exposed to ATRA, the transfected cells showed little or statistically insignificant changes in Ara-C sensitivity after ATRA treatment. We conclude that data from the transfectants provides evidence that expression of bcl-2 is a determinant of sensitivity to Ara-C and H2O2; and that the effect of ATRA on sensitivity requires the presence of bcl-2 genes in association with regulatory elements.

Acute Disease↗

PubMed1995

Differential expression of a basic helix-loop-helix phosphoprotein gene, G0S8, in acute leukemia and localization to human chromosome 1q31.

A basic helix-loop-helix phosphoprotein gene, G0S8, was recently isolated by differential screening of cDNA from human blood mononuclear cells stimulated with a T cell mitogen and cycloheximide. In this study, G0S8 expression was examined in normal and malignant hematopoietic cells by Northern blot analysis and reverse transcription-polymerase chain reaction (RT-PCR). G0S8 expression was observed in most fresh samples of acute myelogenous leukemia (AML) (28/30) and most cases of adult acute lymphoblastic leukemia (ALL) (9/11) regardless of clinical classification. G0S8 mRNA was also detected in all cases tested of chronic myelogenous leukemia (CML) in blast crisis. However, G0S8 expression was not detected in CML patients in chronic phase, nor in normal bone marrow or other hematopoietic cells. G0S8 has been mapped using fluorescence in situ hybridization (FISH) to human chromosome 1q31, the same site reported for the B cell homolog BL34/1R20 and within a region implicated in the development of hematological malignancies. The consistent observation of G0S8 mRNA in patient samples of acute leukemia suggests that G0S8 expression may either play a role in leukemogenesis or represent a common consequence of dysregulated growth.

Amino Acid Sequence↗

PubMed1995

Mechanism of cytosine arabinoside toxicity to the blast cells of acute myeloblastic leukemia: involvement of free radicals.

Retinoic acid and hydrocortisone (HC) have been shown to regulate the drug sensitivity of the blast cells of acute myeloblastic leukemia (AML). We asked if the proto-oncogene bcl-2 played a role in this regulation. As target cells we used the continuous lines, OCI/AML-1, OCI/AML-2 or OCI/AML-5; expression of bcl-2 can be detected by Northern analysis of RNA from OCI/AML-2 or OCI/AML-5 cells; bcl-2 expression can be found in OCI/AML-1 cells only by using RT-PCR. Exposure of OCI/AML-2 or OCI/AML-5 cells to retinoic acid (all-trans retinoic acid, ATRA) led to a down-regulation of bcl-2 expression that was first seen after 2 h of exposure and was complete after a day. The down-regulation could be prevented by exposing the cells to ara-C either before or after ATRA; decrease in bcl-2 protein was moderate and only obvious after 36 h of ATRA treatment. Nuclear run-on experiments provided evidence that bcl-2 down-regulation was occurring at transcriptional and post-translational levels. Since bcl-2 is considered to have anti-oxidant activity, we tested the sensitivity of the three cell lines to H2O2; we found that OCI/AML-1, the line with very low bcl-2 expression, was a 100-fold more H2O2-sensitive than OCI/AML-2 or OCI/AML-5, where bcl-2 expression can be detected readily. We then asked if H2O2 sensitivity could be regulated. We found that exposure of cells to HC before H2O2 was protective while ATRA after peroxide treatment increased killing; this is the same pattern of regulation observed when AML blasts are exposed to HC before, or ATRA after ara-C. Finally, we asked whether N-acetylcysteine (NAC), a known radical scavenger would protect cells against ara-C killing. Significant protection was observed when NAC was given before drug, but not if given after drug. NAC protection against ara-C killing was seen for OCI/AML-1 and 2 cells, but not for OCI/AML-5 cells. We interpret the results as follows: ara-C kills cells in two ways: first, directly, by incorporation into DNA and chain termination; second, indirectly, by inducing the production of toxic radicals. Bcl-2 reduces the oxidant activity of such radicals, and is protective. ATRA regulates ara-C toxicity by its action on bcl-2. Left unexplained are the action of HC, which does not affect bcl-2 expression and the mechanism by which ara-C prevents down-regulation of bcl-2 by ATRA.

Acetylcysteine↗

PubMed1994

Expression of the Kit and KitA receptor isoforms in human acute myelogenous leukemia.

Genetic and biologic evidence suggests that the Kit receptor tyrosine kinase is important in early events in hematopoietic stem cell differentiation. Two naturally occurring isoforms of the Kit receptor, termed Kit and KitA, were originally described in mouse cells and, subsequently, in human cells. These isoforms differ by the presence (KitA) or absence (Kit) of four amino acids (Gly-Asn-Asn-Lys) that lie immediately outside the transmembrane domain. RNase protection was used to measure the levels of Kit and KitA mRNA in normal bone marrow and the blast cells from individuals with acute myelogenous leukemia (AML). Although both isoforms were present in all the AML samples tested, there was considerable heterogeneity in the relative levels of the two transcripts, with Kit to KitA RNA ratios varying from as low as 1.3 to as high as 12. In contrast, the ratio of Kit to KitA transcripts in normal bone marrow was tightly clustered between 4.4 and 5.5. Because alterations in the relative levels of expression of Kit and KitA may affect the ability of a cell to respond to the Kit ligand, Steel factor, we examined the Kit/KitA RNA ratio in AML patients that differed with respect to a number of diagnostic, prognostic, and biologic parameters. The relative levels of Kit to KitA RNA was independent of French-American-British subtype, response to therapy, and primary and secondary plating efficiencies in vitro. Thus, these data suggest that the relative levels of the two isoforms of the Kit receptor in AML are not associated with any obvious biologic or clinical parameters and, therefore, may reflect naturally occurring changes in splicing mechanisms as stem cells differentiate.

Amino Acid Sequence↗

PubMed1994

Alterations of p53 and c-myc in the clonal evolution of malignant lymphoma.

We derived the lymphoma cell lines OCI-Ly 13.1 and OCI-Ly 13.2 from a patient with non-Hodgkin's lymphoma at the time of presentation and during chemotherapy-resistant relapse. These lines were of T-cell phenotype and contained the identical T-cell receptor beta-chain rearrangement, indicating that both lines were members of the same malignant clone. The lines differed in their growth characteristics; OCI-Ly 13.1 grew slowly and required growth factors for colony formation, whereas OCI-Ly 13.2 grew rapidly and formed colonies without addition of growth factors. To test whether or not these biologic differences were associated with specific genetic changes, we evaluated the status of the c-myc and p53 genes of both cell lines. The p53 and c-myc genes of OCI-Ly 13.1 were in germline configuration and produced normal-sized transcripts. The p53 protein expressed in OCI-Ly 13.1 was recognized by the anti-p53 monoclonal antibody, PAb240, indicating a conformation typical of p53 proteins expressed by p53 alleles containing a missense mutation. However, sequencing studies of the entire p53 coding region did not reveal any point mutations. In contrast, the cell line OCI-Ly 13.2 contained structural abnormalities of both the c-myc and p53 genes. In addition, one of the p53 alleles was lost as determined by a cDNA probe for the p53 gene (17p 13.1) and the YNZ22.1 probe (17p 13.3). These changes resulted in the absence of p53 protein and mRNA in OCI-Ly 13.2 as detected by immunoprecipitation and Northern blot analysis, respectively. They may be a reflection of disease progression and may be associated with the altered behavior of the malignant cell population within the patient and in vitro.

Base Sequence↗

PubMed1994

Modulation of growth factor receptors on acute myeloblastic leukemia cells by retinoic acid.

The effects of retinoic acid (RA) on the proliferation of acute myeloblastic leukemia (AML) cells were studied. AML samples were divided into three groups. Namely, RA stimulated blast colony formation by AML samples in group A and inhibited that by the samples in group B, regardless of added growth factors. For the samples in group C, RA inhibited the colonies formed by granulocyte colony-stimulating factor (G-CSF) but stimulated those by granulocyte macrophage CSF (GM-CSF). To investigate the mechanism involved, the effects of RA on growth factor receptors on AML cells were examined by flow cytometry using fluorolabeled ligands. For the samples in groups A and B, RA affected neither G-CSF receptor (GR) nor GM-CSF receptor (GMR). For the samples in group C, exposure to 10(-7) M RA for 1 day clearly increased GMR, but did not affect GR. This finding supports the hypothesis that the increase of GMR is one of the causes of the stimulative effects of RA on cells cultured with GM-CSF in group C.

Cell Division↗

PubMed1994

Determination of IL6, IL1, and IL4 in the plasma of patients with multiple myeloma.

Interleukin 6 (IL6) plasma levels were measured in 63 patients with multiple myeloma and 8 individuals with benign monoclonal gammopathy. 15 of these 71 samples showed by an enzyme linked immunosorbent assay (ELISA) detectable levels that ranged from 5 to 107 pg of IL6/ml. The IL6 levels of patients with multiple myeloma did not differ significantly from those of normal individuals (N = 25, range 5-27 pg IL6/ml) (Student's t-test, p = 0.295). The samples were negative for IL4; 3 were found positive for IL1 beta. A correlation between IL6, IL4 and IL1 beta levels and disease status was not observed for this group of patients.

Enzyme-Linked Immunosorbent Assay↗

PubMed1994

Fluorescence-labeling of nicks in DNA from leukemic blast cells as a measure of damage following cytosine arabinoside. Application to the study of regulated drug sensitivity.

Damage to DNA can be assessed using a technique for labeling nicks in DNA by incubating paraformaldehyde-fixed cells in a mixture of biotin-labeled dUTP, dATP with dNTP and DNA polymerase I. The addition of labeled nucleotides can then be identified by fluorescence by their reaction with streptavidin. We have used this method to examine damage to the DNA of OCI/AML-2 cells caused by cytosine arabinoside (ara-C) and the effects of hydrocortisone and retinoic acid on this damage (regulated drug sensitivity). Concurrent measurements of clonogenic cells were used to allow a comparison of damage as shown by labeled nicks in DNA with loss of colony-forming capacity. Both methods gave comparable ara-C dose-response curves, for cells incubated with the drug for 24 h. Both methods showed that exposure of OCI/AML-2 cells to hydrocortisone before ara-C greatly reduced the toxicity of the drug; and that retinoic acid given after ara-C increased both its lethal effects on colony formation and the extent of DNA damage as assessed by labeled nicks. Clonogenic assays required for colony formation are not readily adapted to the study of development and repair of damage. The labeled nick assay is suitable for such kinetic studies. OCI/AML-2 cells were exposed in suspension to either hydrocortisone before ara-C or retinoic acid after ara-C. At 24 h intervals thereafter, cells were harvested, assayed by both methods, and recultured after dilution to the original cell concentration. In cultures exposed only to ara-C (controls), the number of cells with labeled nicks increased during the first 24 h and cells with damaged DNA could be detected for 48-72 h, depending on the ara-C dose in spite of the dilution at each passage. OCI/AML-2 cells exposed to hydrocortisone before drug showed fewer nick-labeled cells than controls at the first observation and damaged cells rapidly disappeared from the population with increasing time. For cells treated with retinoic acid after ara-C, the nick-labeled cell population was greater than controls and remained greater throughout subsequent observations. We propose that in the control cultures, sublethal damage either became lethal with time and was seen as increased numbers of cells with damaged DNA, or alternatively, sublethal damage was repaired. From this point of view we consider that hydrocortisone promotes repair of sublethal damage while retinoic acid inhibits repair.

Cytarabine↗

PubMed1994

Regulation by retinoic acid and hydrocortisone of the anthracycline sensitivity of blast cells of acute myeloblastic leukemia.

The experiments reported here continue the study of regulated drug sensitivity by extending the observations to anthracyclines. Previous work has shown that hydrocortisone (HC) protects AML blast stem cells from the lethal effects of cytosine arabinoside (ara-C) while retinoic acid (ATRA) increases ara-C sensitivity; further mechanisms of regulation of ara-C sensitivity might include increase or decrease in repair of sublethal damage. Anthracycline dose-response curves are characterized by an initial shoulder, followed by exponential decrease in survival with increasing dose. The shoulder portion of such curves may indicate the accumulation of sublethal damage. We used two assays to look for evidence of regulation of anthracycline sensitivity by HC or ATRA; the clonogenic assay for blast stem cells detects drug effects on this crucial population, but only after several days on incubation, during which time repair might occur. Measurements of nicks in DNA show damage in the bulk population of cells, but these can be detected very soon after exposure to drug. Both methods showed the HC protected cells in two continuous cell lines (OCI/AML-2 and OCI/AML-5) while ATRA made the cells more sensitive. Blast cells freshly-obtained from six AML patients were also tested. Both assays showed HC protection and ATRA sensitization in three populations. The clonogenic assay detected both effects in cells from a fourth patient; the nicked DNA assay confirmed both effects in a fifth patient, where the results of the clonogenic assay did not reach statistical significance. Neither ATRA nor HC influenced the sensitivity of blasts from a sixth patient; but these cells were highly resistant to drug. Kinetic studies showed that damage persisted longer after treatment with anthracyclines than with ara-C. OCI/AML-2 cells treated with HC before drug accumulated fewer cells with nicked DNA after daunorubicin (DNR). Cells exposed to ATRA after DNR showed increased toxicity in kinetic experiments. We conclude that sensitivity to anthracyclines may be regulated by ligands for steroid receptors. Furthermore, since growth factors do not regulate anthracyclines' sensitivity, different mechanisms may be operative for the action of ligands for cell surface receptors. Finally, we suggest that retinoic acid might be considered for inclusion in standard anthracycline/ara-C regimens for the treatment of AML.

Adult↗

PubMed1994

Minimal residual disease in bone marrow transplant recipients with chronic myeloid leukemia.

Bone marrow samples of 70 transplant recipients with CML were studied by Southern blot analysis and RT-PCR using a two-step procedure with nested primers. Twenty-two patients were studied once and 48 were assessed on multiple occasions. All patients remained in a hematological remission during the study. The time of follow-up after the transplant ranged from 2 to 144 months with a median of 42 months. Thirty-nine patients (56%) were negative by RT-PCR and Southern blot studies at the time of their last evaluation. The proportion of RT-PCR negative patients increased with the duration of follow-up after the transplant; 36% of patients were RT-PCR negative after 1 year compared with 60% after 2 years and 78% after > or = 5 years. Patients maintained on immunosuppression had a higher probability of remaining RT-PCR positive. Age, sex, time from diagnosis to BMT, as well as acute and chronic GVHD did not influence the RT-PCR status. The majority of patients studied on multiple occasions demonstrated a stable RT-PCR and Southern blot pattern. Some showed uni- or multi-directional transitions. However, none of the patients studied progressed to a hematological relapse. RT-PCR studies on colonies grown from RT-PCR positive. Southern blot negative patients confirmed that some of the clonogenic progenitors are able to produce BCR/ABL transcripts.

Adolescent↗

PubMed1993

Relationship between c-Kit expression and proliferation in acute myeloblastic leukemia cell lines.

We have proposed that the transmembrane receptor encoded by the c-Kit protooncogene and its ligand play an important role in regulating the proliferation of blasts cells in acute myeloblastic leukemia (AML). To test this hypothesis, immunobeads were used to separate blasts from three Kit-expression positive cell lines into strongly Kit-protein positive and weakly Kit-protein positive fractions. The strongly positive fraction had greater proliferative potential than the weakly positive fraction as assessed both by colony-formation in methylcellulose and growth of clonogenic cells in suspension. The reproducibility of the percentage of each blast population found in the strongly and weakly positive fractions provided evidence that Kit-protein expression is regulated. Kinetic experiments provided evidence for reversible transitions between strong and weak Kit protein expression. Thus regulated expression of the Kit receptor may be a mechanism for controlling blast cell growth in culture.

Animals↗