Biomedical subjects
M Prokocimer
Publications and source records attributed to M Prokocimer.
Stable methylation patterns of MYC and other genes regulated during terminal myeloid differentiation.
Changes in DNA methylation are often associated with the modulation of gene expression. The DNA methylation patterns of six genes whose expression is regulated with terminal myeloid maturation were examined in HL-60 cells before and after their differentiation towards granulocytes. Methylation patterns were stable in five of these genes even with strong up- or down-regulation of mRNA levels. A somatic hybrid cell formed from HL-60 and B-lymphoid cells was studied for mRNA expression and DNA methylation of several of these genes. The pattern of methylation of the genes studied in the hybrid cells was a chimera of the parental patterns. These data suggest that changes in DNA methylation may not be necessary for the modulation of expression of many genes during terminal myeloid differentiation. Furthermore, somatic hybrid cell experiments suggest that specific DNA methylation patterns from both parental cells are inherited in a chimeric pattern by the hybrid and are independent of gene expression.
Establishment of a human T-acute lymphoblastic leukemia cell line with a (16;20) chromosome translocation.
A new T-cell line, Loucy, was established from the peripheral blood of a patient with T-cell acute lymphoblastic leukemia (T-ALL). The surface marker analysis of the cell line is OKT3+, OKT4+, THB4+, J5 +/-, OKT6-, TdT-, and HLA-DR-, indicating stage IV in T-cell lineage. Karyotype analysis revealed 45,X,5q-,t(16;20)(p12;q13). The translocation between chromosomes 16 and 20 has not been previously detected in ALL. This cell line may be of value in evaluating the role of t(16;20) in the etiology of T-ALL.
Systemic sclerosis and chronic lymphocytic leukaemia.
The association between the CREST (calcinosis, Raynaud phenomena, oesophageal hypomotility, sclerodactyly and telangiectasia, variant of systemic sclerosis and chronic lymphocytic leukaemia is described in three patients. The present description raises the possibility that the association of chronic lymphocytic leukaemia and systemic sclerosis is non-random.
Rearrangements in the p53 gene in Philadelphia chromosome positive chronic myelogenous leukemia.
Molecular structural analysis of the p53 gene in patients with Philadelphia chromosome-positive chronic myelogenous leukemia (CML) indicates a significant incidence of gene rearrangements in patients at either accelerated phase or blastic crisis. Southern blot analysis of genomic DNA hybridizing with either genomic or cDNA p53 specific probes indicated that 30% of the CML patients at blastic crisis phase exhibited rearrangements, mostly mapping downstream to the first non-coding exon. This is compatible with the observation that the progression of CML from the chronic to the acute phase involves frequent aberrations in chromosome 17, to which the p53 oncogene has been mapped. Therefore, we suggest that one of the pathways of development of CML to the acute phase is associated with aberrations in the p53 nuclear oncogene.
Analysis of BCR-ABL mRNA in chronic myelogenous leukemia patients and identification of a new BCR-related sequence in human DNA.
The Philadelphia chromosome is present in more than 95% of chronic myelogenous leukemia patients and in up to 25% of patients with acute lymphocytic leukemia. The major consequence of the aberration is the fusion of the ABL and BCR genes. The position of the breakpoint on chromosome 22 determines which species of the potential three fused mRNAs and proteins will be synthesized. We have used the polymerase chain reaction (PCR) to detect these mRNAs in 53 patients and cell lines and found that around 20% contain simultaneously two BCR-ABL mRNAs, presumably due to a process of alternative splicing. The results also indicate that most patients in lymphocytic blast crisis of CML contain the mRNA in which bcr exon 2 is linked to ABL exon II. Finally, we identified, cloned, and characterized a BCR-related sequence that originated from mRNA.
Recombinant interferon alpha-C for advanced hairy cell leukemia. An Israeli multicenter study.
Thirteen patients with advanced hairy cell leukemia were treated with a new subspecies of interferon (IFN): recombinant IFN (rIFN)-alpha-C. The timing of the peripheral hematologic remission of individual blood elements was similar to that reported for other interferons, and 60% of patients attained a complete peripheral hematologic remission at 9 months. Two patients relapsed despite a good initial response to IFN. No anti-IFN antibodies could be detected in their sera. In vitro studies of colony formation from the peripheral blood of all responding patients showed that rIFN-alpha-C did not inhibit the growth of colonies but favorably affected the maturation of their elements towards monocytes, granulocytes, and erythroid elements. The relapsing patient examined initially experienced a similar beneficial in vitro response which paralleled his in vivo improvement. During relapse, rIFN-alpha-C inhibited both the colony formation and the myelomonocytic differentiation in the in vitro cultures. These findings may suggest that the acquired resistance to IFN in our patient could be due either to an acquired stem cell maturation arrest in response to IFN or to emergence of a new clone indifferent to IFN-alpha-C differentiation effect.
Autoimmunity in chronic lymphocytic leukaemia.
Seventy-nine patients with chronic lymphocytic leukaemia were evaluated for the presence of autoimmune diseases and autoantibodies. One patient has polymyositis and two additional patients presented with features suggestive of pernicious anaemia and chronic active hepatitis. The Coombs' direct test was positive in 7% and immune thrombocytopenia was present in 8.1% of patients. Five (7%) patients had M-protein in the serum. No increased frequency of other autoantibodies was noted in our study group. We conclude that the propensity to develop antibodies is restricted only to the haematopoietic system and that there is no increased frequency of non-haematological autoimmune diseases in chronic lymphatic leukaemia.
Hypercalcemia complicating myelomonoblastic transformation of chronic myeloid leukemia.
We present a rare case of hypercalcemia complicating myelomonoblastic transformation of CML and comment upon the pathogenesis and management of the hypercalcemia at this stage of the disease.
Interaction of soybean agglutinin with human leukemia-lymphoma lines at various stages of differentiation.
Human leukemia-lymphoma cell lines reflecting hematopoietic clones at various stages of differentiation were examined for reactivity with soybean agglutinin (SBA). The binding and redistribution pattern of soybean surface receptors was determined with fluorescein-isothiocyanate conjugated SBA (F-SBA) by ultraviolet microscopy, and with a fluorescent activated cell sorter (FACS). The results indicate that there is a correlation between SBA labelling--distribution and the stage of lymphoid cell differentiation. The SBA labelling on the membrane of null lines was undetectable by U.V. microscopy and flow cytometry. A gradual increase in SBA labelling correlating with the stage of differentiation was observed on cell lines of both B and T origin. However the maximal fluorescence intensity of the T lines was lower than the B lines. The redistribution pattern of SBA on the membrane of T lines was rings and mild patches, whereas that on the B lines was moderate to large patches. The reactivity of the lymphoid lines with SBA was not affected by growth conditions. The binding of SBA to normal lymphoblastoid lines was generally low and the fluorescence intensity weak. The reactivity of these lines with SBA was not associated with their origin or "age". It is suggested that the differences in the reactivity of SBA with human hematopoietic lines at various stages of maturation may be of value in future understanding the differences in structure and function of the surface membrane between normal and malignant cells, and the relation to normal and abnormal cell differentiation.
Primary malignant neoplasms associated with chronic lymphocytic leukaemia.
The relationship between chronic lymphocytic leukaemia (CLL) and primary malignant neoplasms was evaluated using data from the Hematology Division in Beilinson Medical Center and the Israel Cancer Registry. The study population consisted of 81 patients diagnosed between 1962 and 1984. A total of 16 patients were found to have 21 malignant neoplasms in addition to their CLL. Excluding patients with nonmelanoma skin tumours, a 1.7 increased risk (statistically not significant) for developing second malignant neoplasms in CLL patients was detected. The only tumour which occurred significantly more than expected subsequent to CLL diagnosis was brain cancer. The coexistence of multiple cancers in the same patient was diagnosed in four of the patients. The results of this study further support the hypothesis that patients with CLL are prone to develop second neoplasms.
Molecular basis for heterogeneity of the human p53 protein.
The human p53 tumor antigen comprises several physically distinct proteins. Two p53 proteins, separable by polyacrylamide gel electrophoresis, are expressed by the human transformed cell line SV-80. The individual cDNAs which code for these proteins were isolated and constructed into the SP6 transcription vector. The proteins encoded by these clones were identified by in vitro transcription with the SP6 vector and translation in a cell-free system. p53-H-1 and p53-H-19 cDNA clones code for the faster- and slower-migrating p53 protein species, respectively, of SV-80. The in vitro-expressed proteins of p53-H-1 and p53-H-19 had the same antigenic determinants and were structurally indistinguishable from their in vivo counterparts. By expressing defined restricted cDNA fragments in vitro, the region of heterogeneity between the respective cDNAs was located at the 5' end of the cDNAs. Exchanging the 5' fragments of interest and expressing the chimeric clones in vitro confirmed that the DNA heterogeneity was responsible for the difference in the electrophoretic mobility of these proteins. The sequences of the two cDNAs revealed a single base pair difference (G versus C) in the coding region of the clones. This sequence difference resulted in an arginine being coded for in clone p53-H-1 and a proline being coded for at the equivalent position in clone p53-H-19. This variation accounted for the change in the electrophoretic mobility of the individual p53 protein species.
Induction of granulocyte differentiation in a human leukemia cell line does not require cell division.
The human promyelocytic cell line, known as HL-60, provides a model for the study of myeloid differentiation. The HL-60 can differentiate to granulocyte-like cells in the presence of dimethylsulfoxide (DMSO). We investigated the requirement for DNA synthesis in the myeloid differentiation process and found that induction of differentiation of HL-60 cells did not require 3H-thymidine incorporation as judged by autoradiography. The results indicate that the triggering of granulocyte differentiation of HL-60 can occur in the absence of DNA synthesis.
Expression of p53 in human leukemia and lymphoma.
Analysis of fresh human tumors have indicated that patients with B type lymphoproliferative diseases and the majority of patients with acute lymphoblastic leukemia (ALL) express elevated levels of p53 production. It is suggested that in these human malignancies, p53 may provide a novel tool for monitoring cancer activity. Conversely, p53 is not expressed in acute myeloid leukemias, myeloproliferative diseases, or myeloid leukemic cell lines. Analysis of the p53 gene structure indicated the existence of similar patterns of p53 restriction fragments in producer and nonproducer cells, which suggests that the p53 gene is not altered in the latter. However, in one case of acute promyelocytic leukemia (APL), we have observed a rearrangement in the p53 gene. Karyotype analysis has indicated that these APL cells do not contain the typical 15;17 translocation. In other APL patients who exhibit a 15;17 translocation, we found no genomic changes of the p53, suggesting that the p53 gene, which was recently mapped to the short arm of chromosome 17 in the human, is not structurally related to the typical chromosomal break point found in the long arm of chromosome 17 of APL patients.
Study of differentiation of fresh myelogenous leukemic cells by compounds that induce a human promyelocytic leukemic line (HL-60) to differentiate.
The human promyelocytic leukemia cell line known as HL-60 can be triggered to mature to functional granulocytes and/or macrophages after exposure to a variety of compounds. The findings have generated enthusiasm for possible therapy of leukemia using compounds that induce leukemic cell differentiation. We investigated whether five compounds known to trigger HL-60 differentiation to granulocytes could trigger the maturation of blast cells from 12 patients with myelogenous leukemia. Maturation was judged by morphology, superoxide production, phagocytosis, expression of Fc receptors, and development of alpha-napthyl acetate esterase activity. The blast cells from most patients showed little morphological, histological or functional maturation after exposure to the various compounds as compared to the blast cells cultured without the compounds. Actinomycin was able to induce significant maturation of leukemic cells of some patients when maturation was analyzed by several statistical methods. Our study suggests that many compounds which trigger differentiation of promyelocytic leukemia cells may not trigger differentiation of less mature myeloid leukemic cells.
Hemophagocytosis simulating malignant histiocytosis: a terminal event of the myelodysplastic syndrome.
We describe a patient with hemophagocytic syndrome resembling malignant histiocytosis which was complicating myelodysplastic disease of 3 years duration. Detailed morphological and ultrastructural studies indicate that the histiocytic component did not demonstrate features of malignancy. A review of other known malignancies ending up in the hemophagocytic syndrome is given, and the significance of this syndrome is discussed.
Multivariate analysis of prognostic factors in chronic lymphocytic leukemia.
Prognostic factors in a group of 90 patients with chronic lymphocytic leukemia were studied by methods of survival analysis. The relationship between survival and a set of demographic, clinical and laboratory variables, and identification of subsets of variables that are associated with survival, was tested by multivariate analysis, which is based upon Cox proportional hazards regression models in a stepwise procedure. Six variables showed significant correlation with survival: lymph node enlargement, splenomegaly, hepatomegaly, increased percentage (greater than 80%) of lymphocytes, hyperuricemia, and anemia. Stepwise analysis showed that the number of coexistent risk factors was a better predictor of survival than any single risk marker (P less than 0.001). Median survival of patients with 0 or 1 risk marker was 120 months; with 2 or 3, 96 months; with 4, 36 months; and with 5 or 6, only 24 months. Comparison of staging by number of risk markers with staging of the same patients by the Rai system showed a significant trend of decreasing survival with increasing number of risk markers within the same Rai stage. Staging by the number of coexistent risk markers is a simple and readily available method, which may complement existing methods to provide a more accurate assessment of prognosis in patients with chronic lymphocytic leukemia.
Concanavalin A receptors on the surface membrane of lymphocytes from patients with acute leukemia.
Peripheral blood mononuclear cells (PBM) isolated from 23 patients with acute lymphoblastic leukemia (ALL) and 24 with acute non-lymphoblastic leukemia (ANLL) were studied for binding and mobility of Concanavalin A (Con A) receptors, using fluorescent Con A (F-Con-A). The cap forming ability of PBM from all patients was 18.7 (+/- 9.3%) and 18.9 (+/- 9.9%) for ANLL patients at the time of diagnosis or during relapse. During clinical complete remission the cap forming ability of the PBM did not change significantly. No correlation was observed between the percentage of blasts present in the peripheral blood at the time of examination and the extent of cap formation, for both types of leukemia. The pattern of F-Con-A binding to PBM in ANLL patients was different compared to that seen in ALL. In ANLL, the fluorescent stain was concentrated in a round body on the cell ("button form") after binding to the membrane, while the rest of the cell showed almost no fluorescence. The present results indicate that PBM cells from patients with acute leukemia are characterized by a high degree of Con-A receptor mobility.