Hematopoietic Neoplasia Research Articles

Leukemia-lymphoma cell lines as model systems for hematopoietic research

Continuous human leukemia-lymphoma (LL) cell lines comprise a rich self-renewing resource of accessible and manipulable living cells which has illuminated the pathophysiology of hematopoietic tumors as well as basic cell biology. The major key advantages of continuous cell lines are the unlimited supply and worldwide availability of identical cell material and their cryopreservation. LL cell lines are characterized generally by monoclonal origin and differentiation arrest, sustained proliferation in vitro with preservation of most cellular features, and specific genetic alterations. The most practical classification of LL cell lines assigns them to one of the physiologically occurring cell lineages, based on their immunophenotype, genotype and functional features. Truly malignant cell lines may be distinguished from Epstein-Barr virus (EBV)-immortalized normal cells, using various operational and conceptual parameters. The characterization and publication of new LL cell lines provides important and informative core data which, by opening new avenues for investigation, have become ubiquitous powerful research tools that are available to every investigator by reference cell repositories. There is a need in the scientific community for clean and authenticated LL cell lines to which every scientist has access as offered by these institutionalized public cell line banks. A list of the most useful, robust and freely available reference cell lines is proposed in this review. Clearly, studies of LL cell lines have provided seminal insights into the biology of hematopoietic neoplasia.

Serous retinal detachment in a case with chronic lymphocytic leukemia: no response to systemic and local treatment

Leukemias are systemic hematopoietic neoplasias and not infrequently cause ocular findings. Serous retinal detachment (SRD) is one of these manifestations and even may be the first sign of the underlying leukemia. Here we reported a case with chronic lymphocytic leukemia (CLL) presenting with SRD and discussed the clinical importance and therapeutic options.

Transdifferentiation and nuclear reprogramming in hematopoietic development and neoplasia.

Cell transplantation and tissue regeneration studies indicate a surprisingly broad developmental potential for lineage-committed hematopoietic stem cells (HSCs). Under these conditions HSCs transition into myocytes, neurons, hepatocytes or other types of nonhematopoietic effector cells. Equally impressive is the progression of committed neuronal stem cells (NSCs) to functional blood elements. Although critical cell-of-origin issues remain unresolved, the possibility of lineage switching is strengthened by a few well-controlled examples of cell-type conversion. At the molecular level, switching probably initiates from environmental signals that induce epigenetic modifications, resulting in changes in chromatin configuration. In turn, these changes affect patterns of gene expression that mediate divergent developmental programs. This review examines recent findings in nuclear reprogramming and cell fusion as potential causative mechanisms for transdifferentiation during normal and malignant hematopoiesis.

Molecular diagnosis in hematopathology

The diagnosis of hematopoietic neoplasias has nowadays become more and more refined by molecular cytogenetic and molecular biological techniques. The biological basis of most of these techniques is that (malignant) tumors represent clonal neoplasias that are derived from a single or few progenitor cells, thus imparting a clonal relationship to all daughter cells. Apart from clonality analysis as a marker for neoplastic growth, the presence or absence of certain primary chromosome abnormalities, and the number and the kind of secondary genetic anomalies become increasingly important in the delineation of the biological grade of aggressiveness.

Longitudinal analysis of feline leukemia virus-specific cytotoxic T lymphocytes: correlation with recovery from infection.

Feline leukemia virus (FeLV) is a common naturally occurring gammaretrovirus of domestic cats that is associated with degenerative diseases of the hematopoietic system, immunodeficiency, and neoplasia. Although the majority of cats exposed to FeLV develop a transient infection and recover, a proportion of cats become persistently viremic and many subsequently develop fatal diseases. To define the dominant host immune effector mechanisms responsible for the outcome of infection, we studied the longitudinal changes in FeLV-specific cytotoxic T lymphocytes (CTLs) in a group of naïve cats following oronasal exposure to FeLV. Using (51)Cr release assays to measure ex vivo virus-specific cytotoxicity, the emerging virus-specific CTL response was correlated with modulations in viral burden as assessed by detection of infectious virus, FeLV p27 capsid antigen, and proviral DNA in the blood. High levels of circulating FeLV-specific effector CTLs appeared before virus neutralizing antibodies in cats that recovered from exposure to FeLV. In contrast, persistent viremia was associated with a silencing of virus-specific humoral and cell-mediated host immune effector mechanisms. A single transfer of between 2 x 10(7) and 1 x 10(8) autologous, antigen-activated lymphoblasts was associated with a downmodulation in viral burden in vivo. The results suggest an important role for FeLV-specific CTLs in retroviral immunity and demonstrate the potential to modulate disease outcome by the adoptive transfer of antigen-specific T cells in vivo.

FISH monitoring of 100 courses of human leukemias: The cytogenetic viewpoint

Interphase fluorescence in situ hybridization (I-FISH) analyses were performed from 2 up to 13 times along the course of 100 human leukemias (36 chronic myeloid leukemias, 38 acute myeloblastic leukemias, 17 acute lymphoblastic leukemias, and 9 additional hematopoietic neoplasias) in order to control clonality, evolution, and disappearance of the basic cytogenetic changes. The relevance of these data to confirm clinical remission or to detect minimal residual disease and/or relapse was evaluated. Fifty-four patients were monitored following hematopoietic stem cell transplantation, and 46 cases after chemotherapy. Various chromosome or aberration specific DNA probes were applied for follow-up in the time frame of 1 month up to 13 years. From the cytogenetic point of view, the aim was to determine the power of resolution of the I-FISH technique in the detection of clinically significant changes in the course of disease and its usefulness in daily routine cyto-genetics as compared with classical cytogenetics. In addition, reliability standards of the various DNA probes should be established. In 75 patients with remissions during the entire period of I-FISH monitoring no conspicuous signal constitution was detected. Of 25 relapses or progresses of disease, which were clinically confirmed, 22 were reliably detected by I-FISH, in only 1 case I-FISH monitoring failed to detect the aberrant clone. In 2 patients conventional karyotype analysis confirmed the relapse by detecting complex chromosomal aberrations, but specific probes for I-FISH confirmation were not available. These data suggest that I-FISH analyses in the follow-up of leukemias is a simple and in most cases sufficiently sensitive and highly reliable way of monitoring the outcome of therapy. It may well serve to close the gap between conventional karyotyping and the highly sensitive molecular techniques.

Comparative genomic hybridization-aided unraveling of complex karyotypes in human hematopoietic neoplasias

The information obtained by conventional cytogenetics (CC) in human leukemias is sometimes limited, in particular by complex karyotypes with many marker chromosomes. While CC is restricted to metaphases with a good quality, interphase fluorescence in situ hybridization (I-FISH) is also capable of analyzing specific anomalies in the interphase nuclei. Comparative genomic hybridization (CGH) gives additional information about the imbalanced karyotype changes in the whole genome. The aim of this study was to assess the contribution of CGH to the unraveling of complex GTG karyotypes, which are difficult to evaluate by banding analysis, and to compare these results with those by CC and FISH. Thirteen bone marrow samples and one sample obtained from peripheral blood of 13 leukemia patients were examined by CC, FISH and CGH. The GTG banding analysis showed complex karyotypes with many marker chromosomes. The most frequent abnormalities were numerical and structural aberrations on chromosomes 5 and 7. In 12 of the 14 samples, the CGH analysis was able to detect chromosomal imbalances with losses of material on chromosome 5 and 7 as the most frequent aberrations. In all 14 samples, additional FISH analyses were performed. For most of the studied neoplasias, a close correlation between CC, FISH and CGH data was observed. CGH was considerably helpful in adding additional information to classical karyotyping, if the low quality or number of metaphases was insufficient for a reliable CC analysis. Even in cases where whole chromosome painting could be applied, it added information on the breakpoints of the observed rearrangements. In only 2 of the studied 14 samples, neither CGH nor I-FISH could improve the result of karyotyping. CGH, nevertheless, can be regarded as a powerful additional technique in leukemias with unsuccessful CC, incomplete, or complex karyotypes with many marker chromosomes. A systematic analysis by three techniques such as CC, FISH and CGH guarantees an optimal genetic characterization of the neoplasias.

Selectins (CD62L, CD62P) and megakaryocytic glycoproteins (CD41a, CD42b) mediate megakaryocyte–fibroblast interactions in human bone marrow

Previous in vitro studies are in keeping with the finding that isolated and enriched megakaryocytes attach to bone marrow fibroblasts and generate an increased growth of these cells. This process was assumed to depend on a close spatial relationship between both cell types which supports the paracrine effect of platelet-derived growth factor (PDGF) and transforming growth factor (TGF)-β1. Moreover, adhesion molecules including β1 integrin receptors and fucosylated structures were determined to play an important role in these complex interactions. However, up to now the influence of megakaryocyte expressed glycoproteins CD41a and CD42b in these processes was not investigated. In addition, the role of megakaryocytic CD62P and also of CD62L, both adhesion molecules of the selectin group, could also be of interest. Following isolation and enrichment of bone marrow megakaryocytes and fibroblasts, both cell populations were characterized regarding their expression of these factors by applying immunocytochemical techniques. Additionally, their influence on adhesion of megakaryocytes to fibroblasts as well as fibroblast growth was evaluated by comparative megakaryocyte–fibroblast co-cultures and inhibition studies using specific monoclonal antibodies (mabs). Fibroblast monocultures served as controls. In these experiments, selectin-specific antibodies significantly reduced megakaryocyte attachment to fibroblast feeder layers and fibroblast growth in the co-cultures. The effect of CD41a and CD42b specific antibodies was limited to megakaryocyte-dependent fibroblast growth. These results elucidate the involvement of the selectins CD62P and CD62L in the basal activation of megakaryocytes inducing their attachment to bone marrow fibroblasts. In contrast, the megakaryocyte glycoproteins CD41a and CD42b exert their effect on the megakaryocyte dependent fibroblast growth. Altogether, it is tempting to speculate that the various interactions of these mediators reflect certain steps in the complex pathomechanisms causing the evolution of (reactive) myelofibrosis in hematopoietic neoplasias accompanied by megakaryocytic proliferation.

Hematopoietic neoplasia in C57BL/6 mice exposed to split-dose ionizing radiation and circularly polarized 60 Hz magnetic fields

This study assessed the effect of chronic exposure to a 60 Hz circularly polarized magnetic field on the occurrence of ionizing radiation-induced lymphoma and other hematopoietic neoplasia in mice. Female C57BL/6 mice received lifetime exposure to either a magnetic field flux density of 1.42 mT for 18 h/day, or an ambient magnetic field of 0.13 microT. Beginning on the first day of magnetic field exposure, 1710 mice were treated with one of three levels of split-dose Cobalt-60 gamma-radiation (cumulative 3.0, 4.0 or 5.1 Gy). The remaining 570 mice received sham irradiation treatment. Sections from 10 lymphoid tissues were evaluated histopathologically for hematopoietic neoplasia. The primary statistical analysis used the Poly3 method to compare lymphoma incidences in magnetic field (MF)-exposed and control mice. Secondary analyses used the Cox proportional hazards model to analyze incidence rates for mortality and development of specific types of neoplasia. The mortality incidence rate was increased by ionizing radiation treatment, and all neoplasms were observed sooner in irradiated mice. However, the lifetime incidence of hematopoietic neoplasia was similar in all experimental groups, including those that were not exposed to ionizing radiation. Chronic exposure to MFs did not affect the mortality incidence rates and did not change the relative incidences of hematopoietic neoplasia in mice that received the same ionizing radiation treatment, with the exception of a marginally significant reduced relative risk of 0.97 (P = 0.05) for lymphoblastic lymphoma in mice exposed to a magnetic field and treated with 5.1 Gy. Lymphomas and histiocytic sarcomas were first observed approximately 50 days sooner in mice that were exposed to magnetic fields but not ionizing radiation, although this comparison was not statistically significant and the incidence of hematopoietic neoplasia in these mice was not different from that of mice in the 0 T/0 Gy group.

Hematopoietic neoplasia in C57BL/6 mice exposed to split-dose ionizing radiation and circularly polarized 60 Hz magnetic fields

This study assessed the effect of chronic exposure to a 60 Hz circularly polarized magnetic field on the occurrence of ionizing radiation-induced lymphoma and other hematopoietic neoplasia in mice. Female C57BL/6 mice received lifetime exposure to either a magnetic field flux density of 1.42 mT for 18 h/day, or an ambient magnetic field of 0.13 microT. Beginning on the first day of magnetic field exposure, 1710 mice were treated with one of three levels of split-dose Cobalt-60 gamma-radiation (cumulative 3.0, 4.0 or 5.1 Gy). The remaining 570 mice received sham irradiation treatment. Sections from 10 lymphoid tissues were evaluated histopathologically for hematopoietic neoplasia. The primary statistical analysis used the Poly3 method to compare lymphoma incidences in magnetic field (MF)-exposed and control mice. Secondary analyses used the Cox proportional hazards model to analyze incidence rates for mortality and development of specific types of neoplasia. The mortality incidence rate was increased by ionizing radiation treatment, and all neoplasms were observed sooner in irradiated mice. However, the lifetime incidence of hematopoietic neoplasia was similar in all experimental groups, including those that were not exposed to ionizing radiation. Chronic exposure to MFs did not affect the mortality incidence rates and did not change the relative incidences of hematopoietic neoplasia in mice that received the same ionizing radiation treatment, with the exception of a marginally significant reduced relative risk of 0.97 (P = 0.05) for lymphoblastic lymphoma in mice exposed to a magnetic field and treated with 5.1 Gy. Lymphomas and histiocytic sarcomas were first observed approximately 50 days sooner in mice that were exposed to magnetic fields but not ionizing radiation, although this comparison was not statistically significant and the incidence of hematopoietic neoplasia in these mice was not different from that of mice in the 0 T/0 Gy group.

Lineage-Restricted Expression of Bone Morphogenetic Protein Genes in Human Hematopoietic Cell Lines

ABSTRACT: To explore the possibility that bone morphogenetic proteins (BMPs) are autocrine/paracrine regulators of hematopoietic differentiation and function, we screened a panel of human cell lines encompassing the hematopoietic lineages for expression of members of this family of genes. Expression of BMP-2, BMP-4, BMP-6, BMP-7, Growth and Differentiation Factor-1 (GDF-1), Placental Bone Morphogenetic Protein (PLAB), and Transforming Growth Factor-β3 (TGF-β3) was detected in one or more cell lines. BMP-2, BMP-4, BMP-7, and TGF-β3 expression was also found in normal hematopoietic tissue. Expression of BMP-5 and BMP-8 was not seen. Lineage-restricted patterns of expression were found for BMP-4 (T-lymphoid), BMP-7 (lymphoid), PLAB (macrophage/monocyte), and GDF-1 (myeloid). Expression of BMP-2, GDF-1, and PLAB could be modulated by treatment with differentiating agents. Marked variations in the levels of BMP-4, BMP-7, and PLAB expression were encountered, indicating that disorders in BMP signaling pathways may play a role in the development of hematopoietic neoplasia.

Spectrum of transforming sequences detected by tumorigenicity assay in a large series of human neoplasms.

We here summarize the analysis of 126 DNA samples from patients with hematopoietic neoplasias and solid tumors and from various tumor cell lines that were screened in the tumorigenicity assay. Thirty-eight samples were able to induce tumors after transfection in NIH/3T3 cells and injection into nude mice. Southern-blot analysis with a panel of oncogene probes revealed human ras genes in the vast majority of cases (25 N-ras, 2 K-ras, 1 H-ras) but also activated FGF4, dbl, ret and mas genes respectively. DNA samples from the 6 remaining transfectants were cloned into EMBL-3 phages and screened with a human specific repetitive Alu probe. Direct hybridization of a transfectant cDNA library allowed cloning of the ufo oncogene. Application of the exon-trapping technique to alu-positive phage DNA from the other transfectants enabled us to isolate tre, cot, B-raf, p85beta/HUMORF8 and a novel oncogene.

Lymphoproliferative and Myeloproliferative Disorders

Nomenclature regarding neoplasia of the hematopoietic and lymphoid tissues in the horse is confusing. This article will clarify terminology, and discuss the individual lymphoproliferative and myeloproliferative disorders recognized in the horse. Diagnostic techniques that are useful in cases in which hematopoietic or lymphoid tissue neoplasia are suspected include histochemical staining profiles, bone marrow aspiration, and bone marrow biopsy.

Exposure to 60 Hz magnetic fields and risk of lymphoma in PIM transgenic and TSG-p53 (p53 knockout) mice.

The results of a number of epidemiology studies suggest that exposure to power frequency (50 and 60 Hz) magnetic fields may be a risk factor for hematopoietic neoplasia. To generate experimental data to test this hypothesis, the influence of magnetic field exposure on lymphoma induction was determined in two strains of mice that are genetically predisposed to the disease. PIM mice, which carry the pim-1 oncogene, are highly sensitive to lymphoma induction by N-ethyl-N-nitrosourea (ENU); ENU-treated PIM mice were studied as a 'high incidence' lymphoma model. TSG-p53 (p53 knockout) mice, in which the p53 tumor suppressor gene has been deleted from the germ line, develop lymphoma as an age-related change; hemizygous TSG-p53 mice were studied as a 'low incidence' lymphoma model. Beginning 1 day after a single i.p. injection of 25 mg ENU/kg body wt, groups of 30 PIM mice/sex were exposed for 18.5 h/day to pure, linearly polarized, transient-free 60 Hz magnetic fields at field strengths of 0 (sham control), 0.02, 2.0 or 10.0 Gauss (G). An additional group of 30 PIM mice/sex was exposed intermittently (1 h on, 1 h off) to 10.0 G fields. Groups of 30 TSG-p53 mice/sex were exposed continuously to magnetic field strengths of 0 (sham control) or 10.0 G; TSG-p53 mice received no ENU. Studies were terminated after 23 weeks of magnetic field exposure. Lymphoma incidence in male PIM mice exposed continuously to 10.0 G magnetic fields was significantly reduced from that seen in sex-matched sham controls; survival, lymphoma incidence and lymphoma latency in other groups of PIM mice did not differ from sham controls. Survival and lymphoma incidence in all groups of TSG-p53 mice was 7% or less, regardless of magnetic field exposure regimen. These data do not support the hypothesis that exposure to magnetic fields is a significant risk factor for lymphoid neoplasia in mice with a genetic predisposition to the disease.

Experimental Field Studies withMya arenaria(Bivalvia) on the Induction and Effect of Hematopoietic Neoplasia

A field experiment was conducted from 1991 to 1992 to examine induction and impact of hematopoietic neoplasia on the marine bivalveMya arenariain southeastern Massachusetts. Clams were collected from Little Buttermilk Bay and separated into three size classes (20–29, 30–39, and 40–49 mm shell length) in the laboratory. These sizes span the range of adults found in the population. A random subsample of these clams was taken to estimate disease prevalence at the start of the experiment, and this was found to be ≤10% in all size classes. Remaining clams were assigned randomly to two groups: Control and Treated. “Controls” were injected with filtered seawater, while “Treated” clams were injected with hemocytes extracted from diseased individuals. Injection of diseased hemocytes was performed to increase disease prevalence in the Treated group. Clams were returned to New Bedford Harbor, a more contaminated field location, where hematopoietic neoplasia is more prevalent, in January 1991, and characteristics of both groups were monitored for 555 days. Among Controls, probability of survival was size-dependent, with higher survival rates in larger clams. Treated clams had a lower probability of survival than Controls, and the magnitude of treatment effect increased with size class. The impact on survival was evident after 89 days, but it was first shown to be statistically significant after 189 days. Among Controls, probability of disease was strongly season-dependent, increasing in the large size class from 0.19 in spring to 0.50 in summer. During summer, Treated clams had a higher probability of being diseased than Controls. Among survivors, no significant sublethal effects due to treatment were detected in the field experiment. Experimental manipulation of disease prevalence may be a useful tool in future studies. In addition to results pertaining to disease, this study obtained long-term growth information, by size class, on somatic and reproductive tissue and shell size.

Telomerase activity and cytogenetic changes in chronic myeloid leukemia with disease progression.

Progressive telomere shortening is thought to be important in the regulation of cellular senescence and that the upregulation or reactivation of telomerase activity may be a critical if not rate limiting step in the development of neoplastic cells. To obtain information about telomeres and telomerase activity in hematopoietic neoplasia at various disease stages, we evaluated 54 samples obtained from 41 patients with chronic myeloid leukemia (CML) using a combination of fluorescent-telomeric repeat amplification protocol and an internal telomerase assay standard. The terminal restriction fragment (TRF) lengths in the blast phase was reduced compared to that in the chronic phase (4.53 +/- 0.72 kb vs 6.13 +/- 1.68 kb; P = 0.0005). All samples obtained from CML in the chronic phase (n = 33) had detectable telomerase activity above background, regardless of age. In the blast phase (n = 21), a significant increase of telomerase activity was detected compared to that in the chronic phase (33.84 +/- 37.86% vs 6.08 +/- 3.21; P = 0.016). Among patients in the blastic phase, 50% of patients had moderate to high telomerase activity (>10 relative value), and the remaining patients had telomerase activity higher than that in the normal peripheral blood cells. No significant differences in hematologic findings, duration of chronic phase or blast phase, and telomere length in the blastic phase were noted between these two groups separated by telomerase activity. CML patients with moderate to high telomerase activity had a high frequency of additional cytogenetic changes (P = 0.01).

Harmonization of Animal Clinical Pathology Testing in Toxicity and Safety Studies*

Ten scientific organizations formed a joint international committee to provide expert recommendations for clinical pathology testing of laboratory animal species used in regulated toxicity and safety studies. For repeated-dose studies in rodent species, clinical pathology testing is necessary at study termination. Interim study testing may not be necessary in long-duration studies provided that it has been done in short-duration studies using dose levels not substantially lower than those used in the long-duration studies. For repeated-dose studies in nonrodent species, clinical pathology testing is recommended at study termination and at least once at an earlier interval. For studies of 2 to 6 weeks in duration in nonrodent species, testing is also recommended within 7 days of initiation of dosing, unless it compromises the health of the animals. If a study contains recovery groups, clinical pathology testing at study termination is recommended. The core hematology tests recommended are total leukocyte (white blood cell) count, absolute differential leukocyte count, erythrocyte (red blood cell) count, evaluation of red blood cell morphology, platelet (thrombocyte) count, hemoglobin concentration, hematocrit (or packed cell volume), mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration. In the absence of automated reticulocyte counting capabilities, blood smears from each animal should be prepared for reticulocyte counts. Bone marrow cytology slides should be prepared from each animal at termination. Prothrombin time and activated partial thromboplastin time (or appropriate alternatives) and platelet count are the minimum recommended laboratory tests of hemostasis. The core clinical chemistry tests recommended are glucose, urea nitrogen, creatinine, total protein, albumin, calculated globulin, calcium, sodium, potassium, total cholesterol, and appropriate hepatocellular and hepatobiliary tests. For hepatocellular evaluation, measurement of a minimum of two scientifically appropriate blood tests is recommended, e.g., alanine aminotransferase, aspartate aminotransferase, sorbitol dehydrogenase, glutamate dehydrogenase, or total bile acids. For hepatobiliary evaluation, measurement of a minimum of two scientifically appropriate blood tests is recommended, e.g., alkaline phosphatase, gamma glutamyltransferase, 5′-nucleotidase, total bilirubin, or total bile acids. Urinalysis should be conducted at least once during a study. For routine urinalysis, an overnight collection (approximately 16 hr) is recommended. It is recommended that the core tests should include an assessment of urine appearance (color and turbidity), volume, specific gravity or osmolality, pH, and either the quantitative or semiquantitative determination of total protein and glucose. For carcinogenicity studies, only blood smears should be made from unscheduled sacrifices (decedents) and at study termination to aid in the identification and differentiation of hematopoietic neoplasia.

Harmonization of Animal Clinical Pathology Testing in Toxicity and Safety Studies

Abstract Ten scientific organizations formed a joint international committee to provide expert recommendations for clinical pathology testing of laboratory animal species used in regulated toxicity and safety studies. For repeated-dose studies in rodent species, clinical pathology testing is necessary at study termination. Interim study testing may not be necessary in long-duration studies provided that it has been done in short-duration studies using dose levels not substantially lower than those used in the long-duration studies. For repeated-dose studies in nonrodent species, clinical pathology testing is recommended at study termination and at least once at an earlier interval. For studies of 2 to 6 weeks in duration in nonrodent species, testing is also recommended within 7 days of initiation of dosing, unless it compromises the health of the animals. If a study contains recovery groups, clinical pathology testing at study termination is recommended. The core hematology tests recommended are total leukocyte (white blood cell) count, absolute differential leukocyte count, erythrocyte (red blood cell) count, evaluation of red blood cell morphology, platelet (thrombocyte) count, hemoglobin concentration, hematocrit (or packed cell volume), mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration. In the absence of automated reticulocyte counting capabilities, blood smears from each animal should be prepared for reticulocyte counts. Bone marrow cytology slides should be prepared from each animal at termination. Prothrombin time and activated partial thromboplastin time (or appropriate alternatives) and platelet count are the minimum recommended laboratory tests of hemostasis. The core clinical chemistry tests recommended are glucose, urea nitrogen, creatinine, total protein, albumin, calculated globulin, calcium, sodium, potassium, total cholesterol, and appropriate hepatocellular and hepatobiliary tests. For hepatocellular evaluation, measurement of a minimum of two scientifically appropriate blood tests is recommended, e.g., alanine aminotransferase, aspartate aminotransferase, sorbitol dehydrogenase, glutamate dehydrogenase, or total bile acids. For hepatobiliary evaluation, measurement of a minimum of two scientifically appropriate blood tests is recommended, e.g., alkaline phosphatase, gamma glutamyltransferase, 5′-nucleotidase, total bilirubin, or total bile acids. Urinalysis should be conducted at least once during a study. For routine urinalysis, an overnight collection (approximately 16 hr) is recommended. It is recommended that the core tests should include an assessment of urine appearance (color and turbidity), volume, specific gravity or osmolality, pH, and either the quantitative or semiquantitative determination of total protein and glucose. For carcinogenicity studies, only blood smears should be made from unscheduled sacrifices (decedents) and at study termination to aid in the identification and differentiation of hematopoietic neoplasia.

Genomic Organization and Chromosomal Localization of the DUSP2 Gene, Encoding a MAP Kinase Phosphatase, to Human 2p11.2-q11

The mitogen-induced gene, DUSP2, encodes a nuclear protein, PAC1, that acts as a dual-specific protein phosphatase with stringent substrate specificity for MAP kinase. MAP kinase phosphorylation and consequent enzymatic activation is a central and often obligatory component in signal transduction initiated by growth factor stimulation or resulting from various types of oncogenic transformation. DUSP2 downregulates intracellular signal transduction through the dephosphorylation/inactivation of MAP kinases. To facilitate assessment of the possible role of DUSP2 in growth processes, the genomic structure and chromosomal location of the gene have been determined. DUSP2 has been localized to the pericentromeric region of human chromosome 2 (2p11.2-q1) by analysis of somatic cell hybrids, in situ chromosome hybridization, and genetic linkage analysis using a single-strand conformational polymorphism (SSCP) that has been identified in the 3′ UTR of the gene. No consistent translocations or deletions at this chromosomal site have been reported in hematopoietic neoplasias or other tumors.

Activation of a Drosophila Janus kinase (JAK) causes hematopoietic neoplasia and developmental defects.

In mammals, many cytokines and growth factors stimulate members of the Janus kinase (JAK) family to transduce signals for the proliferation and differentiation of various cell types, particularly in hematopoietic lineages. Mutations in the Drosophila hopscotch (hop) gene, which encodes a JAK, also cause proliferative defects. Loss-of-function alleles result in lethality and underproliferation of diploid tissues of the larva. A dominant gain-of-function allele, Tumorous-lethal (hopTum-l), leads to formation of melanotic tumors and hypertrophy of the larval lymph glands, the hematopoietic organs. We show that a single amino acid change in Hop is associated with the hopTum-l mutation. Overexpression of either wild-type hop or hopTum-l in the larval lymph glands causes melanotic tumors and lymph gland hypertrophy indistinguishable from the original hopTum-l mutation. In addition, overexpression of Hop in other tissues of the larva leads to pattern defects in the adult or to lethality. Finally, overexpression of either hop or hopTum-l in Drosophila cell culture results in tyrosine phosphorylation of Hop protein. However, overexpression of hopTum-l results in greater phosphorylation than overexpression of the wild-type. We conclude that hopTum-l encodes a hyperactive Hop kinase and that overactivity of Hop in lymph glands causes malignant neoplasia of Drosophila blood cells.