ALBERT

Description: Recently, it has been shown that the homozygous deletion of the cyclin- dependent kinase-4 inhibitor (CDK4I;p16) gene, which is mapped to chromosome 9p21, is frequently observed in a wide spectrum of human cancers, including leukemias. Therefore, the CDK4I gene is thought to be a putative tumor-suppressor gene. We report here that both alleles of the CDK4I gene were completely or partially deleted in human leukemia cells derived from both patients and established cell lines. Thirty-seven hematopoietic cell lines and samples from 72 patients with leukemias were examined for homozygous loss of the CDK4I gene locus by Southern blot analysis. We found that a part or the whole of the CDK4I gene was homozygously deleted in 14 of the 37 (38%) cell lines and 4 of 72 (6%) samples from leukemia patients, including 45 with acute myelocytic leukemia, 14 with acute lymphocytic leukemia (ALL), and 13 with chronic myelocytic leukemia in blastic crisis. In the cell lines, the homozygous deletion of the CDK4I gene was detected in a variety of cell lineages, whereas all 4 cases showing the homozygous deletion were confined to ALL. It should be noted that 2 of them had no cytogenetic abnormalities of chromosome 9. Our results suggest that loss of the CDK4I function may contribute to immortalization of human leukemia cells and play a causative role at least in development of human lymphocytic leukemias.

Description: The human erythrocyte membrane protein 4.1 exists in two major electrophoretic forms: 4.1a (80 Kd) and 4.1b (78 Kd). Mass spectrometry and amino acid analysis of the proteolytic peptides derived from carboxyl-terminal regions of these proteins indicate that they differ by deamidation of two aspargine residues at positions 478 and 502. Electrophoretic analysis of carboxyl-terminal peptides has shown that the mobility difference between the two polypeptides is due to the deamidation of Asn502 and not that of Asn478. This observation was confirmed by converting a congener of the protein 4.1b to 4.1a by site- directed mutagenesis of Asn502 to Asp. These results unambiguously demonstrate that deamidation of Asn502 is responsible for conversion of protein 4.1b to 4.1a. Since the conversion of protein 4.1b to 4.1a, under physiological conditions, occurs in a time-dependent manner, our study clearly shows that deamidation is an excellent marker for red blood cell aging.

Description: We investigated the cause of thrombocytosis in 14 patients with tumors producing colony-stimulating factor (CSF). Of the 14 patients, 10 had tumors producing granulocyte-CSF (G-CSF) and 4 had tumors producing granulocyte-macrophage--CSF (GM-CSF). Thrombocytosis of greater than 400 x 10(9)/L was noted in 8 of 10 patients with G-CSF-producing tumors and all 4 patients with GM-CSF-producing tumors. Median peak platelet counts were, respectively, 511 x 10(9)/L (range, 384 to 694 x 10(9)/L) and 579 x 10(9)/L (range, 526 to 910 x 10(9)/L) in patients with tumors producing G-CSF and GM-CSF. In most patients, thrombocytosis declined towards the terminal stage. High interleukin-1 (IL-1) and IL-6 levels were found in addition to CSFs in the plasma or culture supernatants of tumor cells obtained from most patients. In patients with GM-CSF- producing tumors, these specimens had megakaryocyte-CSF (Meg-CSF) activity, which was abolished by anti-GM-CSF antibody. These specimens also had megakaryocyte potentiating (Meg-Pot) activity attributable to both GM-CSF and IL-6. In patients with G-CSF-producing tumors, only Meg- Pot activity due to IL-6 was detected. These results indicate that the thrombocytosis in GM-CSF-producing tumors was caused by both the Meg- CSF activity of GM-CSF and the Meg-Pot activity of IL-6 plus GM-CSF, while that in G-CSF-producing tumors was due to the Meg-Pot activity of IL-6.

Description: The 11q13 breakpoint region of t(11;14) (q13;q32), translocated to the Ig heavy chain locus at 14q32, has been designated as BCL-1 for B-cell leukemia/lymphoma-1, but the nature of the transcriptional unit has long remained unclear. Recently, the PRAD1 gene encoding cyclin D1, isolated from the 11q13 region, was proposed as a candidate BCL-1 gene on the basis of chromosome walking and concordant overexpression of PRAD1 mRNA in cell lines with t(11;14)(q13;q32). We report here molecular analysis of a variant translocation at the BCL-1 locus, t(11;22)(q13;q11), showing juxtaposition of the Ig light chain gene, Ig lambda, to the PRAD1 gene at its 3′ end, resulting in overexpression of PRAD1 mRNA. Because only the PRAD1 gene is present between the Ig heavy chain and light chain gene breakpoints, an identity between BCL-1 and the PRAD1/cyclin D1 gene is strongly indicated.

Description: The MLL gene involved in 11q23 translocations found in the majority of infantile leukemias and some secondary leukemias makes fusion transcripts with genes such as LTG4 (chromosome 4), LTG9 (chromosome 9), and LTG19 (chromosome 19) as a result of reciprocal translocation. We have examined 25 cases of leukemias with 11q23 abnormalities by Southern blot analysis and the reverse transcriptase-polymerase chain reaction (RT-PCR). Using various primer pairs, chimeric mRNAs could be amplified in 6 of 7 leukemias with t(4;11), 6 of 8 leukemias with t(9;11) including secondary leukemia, 8 of 9 leukemias with t(11;19), and 1 with a deletion at 11q23. The chimeric mRNAs were heterogeneous and differential usage of the MLL exons was found, irrespective of the partner chromosomes. Sensitivity studies showed that a single clone with chimeric mRNA in 10(4) to 10(5) cells could be detected. These findings show that the present RT-PCR settings provide a rapid, accurate, and sensitive tool for diagnosing leukemias with 11q23 translocations and for monitoring response to therapy in these patients.

Description: The 11q13 breakpoint region of t(11;14) (q13;q32), translocated to the Ig heavy chain locus at 14q32, has been designated as BCL-1 for B-cell leukemia/lymphoma-1, but the nature of the transcriptional unit has long remained unclear. Recently, the PRAD1 gene encoding cyclin D1, isolated from the 11q13 region, was proposed as a candidate BCL-1 gene on the basis of chromosome walking and concordant overexpression of PRAD1 mRNA in cell lines with t(11;14)(q13;q32). We report here molecular analysis of a variant translocation at the BCL-1 locus, t(11;22)(q13;q11), showing juxtaposition of the Ig light chain gene, Ig lambda, to the PRAD1 gene at its 3′ end, resulting in overexpression of PRAD1 mRNA. Because only the PRAD1 gene is present between the Ig heavy chain and light chain gene breakpoints, an identity between BCL-1 and the PRAD1/cyclin D1 gene is strongly indicated.

Description: B19 human parvovirus is the etiologic agent of transient aplastic crisis. To better understand B19 virus-induced hematopoietic suppression, we studied the host cell range of the virus using in vitro bone marrow cultures. First, B19 virus replication was examined in the presence of various purified cytokines using DNA dot blot analysis. Replication was detected only in erythropoietin-containing cultures. The other cytokines (granulocyte/macrophage colony-stimulating factor [GM-CSF], G-CSF, M-CSF, interleukin-1 [IL-1], IL-2, IL-3, and IL-6) did not support virus replication, indicating the restriction of B19 virus replication to the erythroid cell lineage. Second, hematopoietic progenitor cells were serially assayed in B19-infected and uninfected bone marrow cultures. At initiation, B19 virus infection caused marked and moderate reduction in colony-forming unit erythroid (CFU-E) and burst-forming unit erythroid (BFU-E) numbers, respectively, without affecting CFU-Mix and CFU-GM numbers. Interestingly, the recovery of the erythroid progenitor numbers was observed at a late stage of cultures despite the sustained reduction in erythroblasts. The cells in the bursts derived from such reappearing BFU-E did not contain the virus genome. Although infectious virus was detected in the culture supernatants, the cultured CFU-E harvested at day 5 was relatively resistant to B19 virus infection compared with the CFU-E in fresh bone marrow. These findings suggest that pluripotent stem cells escaped B19 virus infection and restored the erythroid progenitor cells later in infected cultures. We conclude that the target cells of B19 virus are in the erythroid lineage from BFU-E to erythroblasts, with susceptibility to the virus increasing along with differentiation. Furthermore, the suppression of erythropoiesis and the subsequent recovery of the erythroid progenitor numbers in B19-infected liquid cultures may be analogous in part to the clinical features of B19 virus- induced transient aplastic crisis.

Description: We investigated the cause of thrombocytosis in 14 patients with tumors producing colony-stimulating factor (CSF). Of the 14 patients, 10 had tumors producing granulocyte-CSF (G-CSF) and 4 had tumors producing granulocyte-macrophage--CSF (GM-CSF). Thrombocytosis of greater than 400 x 10(9)/L was noted in 8 of 10 patients with G-CSF-producing tumors and all 4 patients with GM-CSF-producing tumors. Median peak platelet counts were, respectively, 511 x 10(9)/L (range, 384 to 694 x 10(9)/L) and 579 x 10(9)/L (range, 526 to 910 x 10(9)/L) in patients with tumors producing G-CSF and GM-CSF. In most patients, thrombocytosis declined towards the terminal stage. High interleukin-1 (IL-1) and IL-6 levels were found in addition to CSFs in the plasma or culture supernatants of tumor cells obtained from most patients. In patients with GM-CSF- producing tumors, these specimens had megakaryocyte-CSF (Meg-CSF) activity, which was abolished by anti-GM-CSF antibody. These specimens also had megakaryocyte potentiating (Meg-Pot) activity attributable to both GM-CSF and IL-6. In patients with G-CSF-producing tumors, only Meg- Pot activity due to IL-6 was detected. These results indicate that the thrombocytosis in GM-CSF-producing tumors was caused by both the Meg- CSF activity of GM-CSF and the Meg-Pot activity of IL-6 plus GM-CSF, while that in G-CSF-producing tumors was due to the Meg-Pot activity of IL-6.

Description: The human erythrocyte membrane protein 4.1 exists in two major electrophoretic forms: 4.1a (80 Kd) and 4.1b (78 Kd). Mass spectrometry and amino acid analysis of the proteolytic peptides derived from carboxyl-terminal regions of these proteins indicate that they differ by deamidation of two aspargine residues at positions 478 and 502. Electrophoretic analysis of carboxyl-terminal peptides has shown that the mobility difference between the two polypeptides is due to the deamidation of Asn502 and not that of Asn478. This observation was confirmed by converting a congener of the protein 4.1b to 4.1a by site- directed mutagenesis of Asn502 to Asp. These results unambiguously demonstrate that deamidation of Asn502 is responsible for conversion of protein 4.1b to 4.1a. Since the conversion of protein 4.1b to 4.1a, under physiological conditions, occurs in a time-dependent manner, our study clearly shows that deamidation is an excellent marker for red blood cell aging.

Description: Recently, it has been shown that the homozygous deletion of the cyclin- dependent kinase-4 inhibitor (CDK4I;p16) gene, which is mapped to chromosome 9p21, is frequently observed in a wide spectrum of human cancers, including leukemias. Therefore, the CDK4I gene is thought to be a putative tumor-suppressor gene. We report here that both alleles of the CDK4I gene were completely or partially deleted in human leukemia cells derived from both patients and established cell lines. Thirty-seven hematopoietic cell lines and samples from 72 patients with leukemias were examined for homozygous loss of the CDK4I gene locus by Southern blot analysis. We found that a part or the whole of the CDK4I gene was homozygously deleted in 14 of the 37 (38%) cell lines and 4 of 72 (6%) samples from leukemia patients, including 45 with acute myelocytic leukemia, 14 with acute lymphocytic leukemia (ALL), and 13 with chronic myelocytic leukemia in blastic crisis. In the cell lines, the homozygous deletion of the CDK4I gene was detected in a variety of cell lineages, whereas all 4 cases showing the homozygous deletion were confined to ALL. It should be noted that 2 of them had no cytogenetic abnormalities of chromosome 9. Our results suggest that loss of the CDK4I function may contribute to immortalization of human leukemia cells and play a causative role at least in development of human lymphocytic leukemias.