Showing papers by "St. Jude Children's Research Hospital published in 1993"

Direct binding of cyclin D to the retinoblastoma gene product (pRb) and pRb phosphorylation by the cyclin D-dependent kinase CDK4.

Abstract: The product (pRb) of the retinoblastoma gene (RB-1) prevents S-phase entry during the cell cycle, and inactivation of this growth-suppressive function is presumed to result from pRb hyperphosphorylation during late G1 phase. Complexes of the cyclin-dependent kinase, cdk4, and each of three different D-type cyclins, assembled in insect Sf9 cells, phosphorylated a pRb fusion protein in vitro at sites identical to those phosphorylated in human T cells. Only D-type cyclins activated cdk4 enzyme activity, whereas cyclins A, B1, and E did not. When Sf9 cells were coinfected with baculovirus vectors encoding human pRb and murine D-type cyclins, cyclins D2 and D3, but not D1, bound pRb with high stoichiometry in intact cells. Introduction of a vector encoding cdk4, together with those expressing pRb and D-type cyclins, induced pRb hyperphosphorylation and dissociation of cyclins D2 and D3, whereas expression of a kinase-defective cdk4 mutant in lieu of the wild-type catalytic subunit yielded ternary complexes. The transcription factor E2F-1 also bound to pRb in insect cells, and coexpression of cyclin D-cdk4 complexes, but neither subunit alone, triggered pRb phosphorylation and prevented its interaction with E2F-1. The D-type cyclins may play dual roles as cdk4 regulatory subunits and as adaptor proteins that physically target active enzyme complexes to particular substrates.

Overexpression of mouse D-type cyclins accelerates G1 phase in rodent fibroblasts.

Abstract: Mammalian D-type cyclins are growth factor-regulated, delayed early response genes that are presumed to control progression through the G1 phase of the cell cycle by governing the activity of cyclin-dependent kinases (cdks). Overexpression of mouse cyclin D1 in serum-stimulated mouse NIH-3T3 and rat-2 fibroblasts increased their rates of G0 to S- and G1- to S-phase transit by several hours, leading to an equivalent contraction of their mean cell generation times. Although such cells remained contact inhibited and anchorage dependent, they manifested a reduced serum requirement for growth and were smaller in size than their normal counterparts. Ectopic expression of cyclin D2 in rodent fibroblasts, either alone or together with exogenous cdk4, shortened their G0- to S-phase interval and reduced their serum dependency, but cyclin D2 alone did not alter cell size significantly. When cells were microinjected during the G1 interval with a monoclonal antibody specifically reactive to cyclin D1, parental rodent fibroblasts and derivatives overexpressing this cyclin were inhibited from entering S phase, but cells injected near the G1/S phase transition were refractory to antibody-induced growth suppression. Thus, cyclin D1, and most likely D2, are rate limiting for G1 progression.

The ornithine decarboxylase gene is a transcriptional target of c-Myc.

Abstract: Constitutive c-myc expression suppresses cell cycle arrest, promotes entry into S phase, and results in the growth factor-independent expression of ornithine decarboxylase (ODC; EC 4.1.1.17). The ODC gene contains a conserved repeat of the Myc binding site, CACGTG, in intron 1. In this report, we demonstrate that c-Myc is a potent transactivator of ODC promoter-reporter gene constructs in fibroblasts that requires the CACGTG repeat. These sites conferred Myc responsiveness on heterologous promoter constructs, suggesting that ODC is regulated by Myc at the level of transcription initiation. Analysis of deletion and point mutants of c-myc revealed that domains required for transactivation of the ODC promoter did not include the leucine zipper of the Myc protein. This suggests that Myc may interact with transcription factors other than Max to transactivate the ODC gene.

The protein tyrosine kinase JAK1 complements defects in interferon-α/β and -γ signal transduction

Abstract: We have produced a cell line which lacks the protein tyrosine kinase JAK1 and is completely defective in interferon response. Complementation of this mutant with JAK1 restored the response, establishing the requirement for JAK1 in both the interferon-α/β and -γ signal transduction pathways. The reciprocal interdependence between JAK1 and Tyk2 activities in the interferon-α pathway, and between JAK1 and JAK2 in the interferon-γ pathway, may reflect a requirement for these kinases in the correct assembly of interferon receptor complexes.

Identification of AML-1 and the (8;21) translocation protein (AML-1/ETO) as sequence-specific DNA-binding proteins: the runt homology domain is required for DNA binding and protein-protein interactions.

Abstract: The AML1 gene on chromosome 21 is disrupted in the (8;21)(q22;q22) translocation associated with acute myelogenous leukemia and encodes a protein with a central 118-amino-acid domain with 69% homology to the Drosophila pair-rule gene, runt. We demonstrate that AML-1 is a DNA-binding protein which specifically interacts with a sequence belonging to the group of enhancer core motifs, TGT/cGGT. Electrophoretic mobility shift analysis of cell extracts identified two AML-1-containing protein-DNA complexes whose electrophoretic mobilities were slower than those of complexes formed with AML-1 produced in vitro. Mixing of in vitro-produced AML-1 with cell extracts prior to gel mobility shift analysis resulted in the formation of higher-order complexes. Deletion mutagenesis of AML-1 revealed that the runt homology domain mediates both sequence-specific DNA binding and protein-protein interactions. The hybrid product, AML-1/ETO, which results from the (8;21) translocation and retains the runt homology domain, both recognizes the AML-1 consensus sequence and interacts with other cellular proteins.

Structure of the murine Jak2 protein-tyrosine kinase and its role in interleukin 3 signal transduction.

Abstract: Interleukin 3 (IL-3) regulates the proliferation and differentiation of hematopoietic cells. Although the IL-3 receptor chains lack kinase catalytic domains, IL-3 induces tyrosine phosphorylation of cellular proteins. To investigate the potential role of the JAK family of protein-tyrosine kinases in IL-3 signal transduction, we have obtained full-length cDNA clones for murine Jak1 and Jak2 protein-tyrosine kinases and prepared antiserum against the predicted proteins. Using antisera against Jak2, we demonstrate that IL-3 stimulation results in the rapid and specific tyrosine phosphorylation of Jak2 and activates its in vitro kinase activity.

In utero rearrangements in the trithorax-related oncogene in infant leukaemias

Abstract: THE majority ( ∼ 75%) of infant acute leukaemias have a reciprocal translocation between chromosome 11q23 and one of several partner chromosomes1. The gene at 1lq23 (named MLL, ALL-1, HRX or HTRX-1; refs 2–6) has been cloned and shares homology with the Drosophila developmental gene trithorax3–5. Rearrangements of this gene (called HRX here) occur in introns and cluster in a region of ∼ 10 kb; individual patients have different breakpoints3–10. Here we describe three pairs of infant twins with concordant leukaemia who each share unique (clonal) but non-constitutive HRX rearrangements in their leukaemic cells, providing evidence that the leukaemogenic event originates in utero and unequivocal support for the intra-placental 'metastasis' hypothesis for leukaemia concordance in twins11.

Fusion of PAX3 to a Member of the Forkhead Family of Transcription Factors in Human Alveolar Rhabdomyosarcoma

Abstract: Alveolar rhabdomyosarcoma, a malignant tumor of skeletal muscle, is characterized by a chromosomal translocation, t(2;13)(q35;q14). This translocation is associated with a structural rearrangement of the gene encoding PAX3, a presumed transcriptional regulator expressed exclusively during embryogenesis. The breakpoint results in a fusion between PAX3 and a gene provisionally named ALV, a novel member of the forkhead family of transcription factors. In PAX3-ALV, the structural integrity of both PAX3 DNA-binding regions, the paired box and homeodomain, are retained while the putative transcriptional activation domain of PAX3 is replaced by the bisected forkhead DNA-binding domain of ALV. Formation of chimeric transcription factors has now been implicated in diverse human tumors of myogenic, hematopoietic, neuroectodermal, and adipocytic origin, suggesting that transcriptional deregulation is a common mechanism of tumorigenesis.

Hematopoietic cell phosphatase associates with the interleukin-3 (IL-3) receptor beta chain and down-regulates IL-3-induced tyrosine phosphorylation and mitogenesis.

Abstract: Hematopoietic cell phosphatase (HCP) is a tyrosine phosphatase with two Src homology 2 (SH2) domains that is predominantly expressed in hematopoietic cells, including cells whose growth is regulated by interleukin-3 (IL-3). The potential effects of HCP on IL-3-induced protein tyrosine phosphorylation and growth regulation were examined to assess the role of HCP in hematopoiesis. Our studies demonstrate that, following ligand binding, HCP specifically associates with the beta chain of the IL-3 receptor through the amino-terminal SH2 domain of HCP, both in vivo and in vitro, and can dephosphorylate the receptor chain in vitro. The effects of increasing or decreasing HCP levels in IL-3-dependent cells were assessed with dexamethasone-inducible constructs containing an HCP cDNA in sense and antisense orientations. Increased HCP levels were found to reduce the levels of IL-3-induced tyrosine phosphorylation of the receptor and to dramatically suppress cell growth. Conversely, decreasing the levels of HCP increased IL-3-induced tyrosine phosphorylation of the receptor and marginally increased growth rate. These results support a role for HCP in the regulation of hematopoietic cell growth and begin to provide a mechanistic explanation for the dramatic effects that the genetic loss of HCP, which occurs in motheaten (me) and viable motheaten (mev) mice, has on hematopoiesis.

Coamplification of the CDK4 Gene with MDM2 and GLI in Human Sarcomas

Abstract: The 34-kilodalton cyclin-dependent kinase, p34cdk4, is a major catalytic subunit of mammalian D-type cyclins, which act during the G1 phase of the cell cycle to enforce the decision of cells to enter S phase. A murine complementary DNA clone was used to clone the cognate human CDK4 gene, which was localized to human chromosome 12, band q13, by fluorescence in situ hybridization. Because this chromosomal band contains the GLI and MDM2 genes, which are frequently amplified in human sarcomas, we analyzed CDK4 copy number and expression in a panel of sarcoma cell lines. An osteosarcoma cell line, OsACL, manifested a 25-fold increased copy number of CDK4 , amplified concordantly with both GLI and MDM2 , whereas a rhabdomyosarcoma cell line, SJRH30, was found to have an amplicon that included CDK4 and GLI but not MDM2. CDK4 mRNA and protein were overexpressed in both cell lines, and nucleotide sequencing analysis indicated that the gene had not sustained mutations. These observations provide the first evidence for amplification of a gene encoding a cell division cycle protein kinase, complement recent data indicating that genes encoding D-type cyclins are targets of chromosomal rearrangement and gene amplification in tumor cells, and suggest that CDK4 amplification might contribute to oncogenesis.

Interferon-induced nuclear signalling by Jak protein tyrosine kinases

Abstract: Interferons IFN-alpha/beta and IFN-gamma act through independent cell-surface receptors, inducing gene expression through tyrosine phosphorylation of cytoplasmic transcription factors . IFN-alpha stimulates phosphorylation and nuclear localization of the 84/91K and 113K subunits of latent ISGF3 (interferon-stimulated gene factor 3), which combine with the 48K DNA-binding subunit to bind regulatory elements of IFN-alpha-responsive genes. IFN-gamma activates p91 alone, inducing IFN-gamma-responsive genes through a distinct DNA element. Genetic complementation studies implicated the tyrosine kinase Tyk2 in IFN-alpha signalling and, more recently, the related Jak2 kinase in IFN-gamma signalling. We now present biochemical evidence for Jak-family kinase involvement in IFN signal transduction. Jak1 was activated in response to IFN-alpha and IFN-gamma; Jak2 responded exclusively to IFN-gamma. Overexpression of either Jak1 or Jak2 stimulated p91 DNA-binding activity and p91-dependent transcription. Overexpression also activated endogenous Jak kinases, suggesting that interactions between Jak kinases are required during interferon signalling.

Interferon gamma inhibits apoptotic cell death in B cell chronic lymphocytic leukemia.

Abstract: The malignant, CD5+ B lymphocytes of B cell chronic lymphocytic leukemia (B-CLL) die by apoptosis in vitro. This is in contrast to the prolonged life span of the leukemic cells in vivo and likely reflects the lack of essential growth factors in the tissue culture medium. We found that interferon gamma (IFN-gamma) inhibits programmed cell death and promotes survival of B-CLL cells in culture. This effect may also be important in vivo: increased serum levels of IFN-gamma, ranging from 60 to > 2,200 pg/ml, were found in 7 of 10 B-CLL samples tested, whereas the sera of 10 healthy individuals did not contain detectable levels of this cytokine (< 20 pg/ml). High levels of IFN-gamma message were detected in RNA from T cell-depleted B-CLL peripheral blood samples by Northern blot analysis. Synthesis of IFN-gamma by B-CLL lymphocytes was confirmed by in situ hybridization and flow cytometry. The majority of B-CLL cells (74-82%) expressed detectable levels of IFN-gamma mRNA, and CD19+ B-CLL cells were labeled with anti-IFN-gamma monoclonal antibodies. These results show that IFN-gamma inhibits programmed cell death in B-CLL cells and suggest that the malignant cells are able to synthesize this cytokine. By delaying apoptosis, IFN-gamma may extend the life span of the malignant cells and thereby contribute to their clonal accumulation.

Association of hematopoietic cell phosphatase with c-Kit after stimulation with c-Kit ligand.

Abstract: Protein tyrosine phosphorylation and dephosphorylation have been implicated in the growth and functional responses of hematopoietic cells. Recent studies have identified a novel protein tyrosine phosphatase, termed hematopoietic cell phosphatase (HCP) or PTP1C, that is predominantly expressed in hematopoietic cells. HCP encodes a cytoplasmic phosphatase that contains two src homology 2 (SH2) domains. Since SH2 domains have been shown to target the association of signal-transducing molecules with activated growth factor receptors containing intrinsic protein kinase activity, we assessed the association of HCP with two hematopoietic growth factor receptors, c-Kit and c-Fms. The results demonstrate that HCP transiently associates with ligand-activated c-Kit but not c-Fms and that this association occurs through the SH2 domains. In both colony-stimulating factor 1- and stem cell factor-stimulated cells, there is a marginal increase in tyrosine phosphorylation of HCP. Lastly, HCP can dephosphorylate autophosphorylated c-Kit and c-Fms in in vitro reactions. The potential role of HCP in stem cell factor signal transduction is discussed.

Magnetic resonance scans should replace biopsies for the diagnosis of diffuse brain stem gliomas: a report from the Children's Cancer Group.

Abstract: CHILDREN'S CANCER GROUP Protocol CCG-9882 was designed to determine the effectiveness of hyperfractionated radiation for the treatment of children and young adults with brain stem gliomas. The study opened for the accrual of patients on September 21, 1988, and was closed on June 30, 1991. Th

Magnetic Resonance Scans Should Replace Biopsies for the Diagnosis of Diffuse Brain Stem Gliomas

Abstract: Children's Cancer Group Protocol CCG-9882 was designed to determine the effectiveness of hyperfractionated radiation for the treatment of children and young adults with brain stem gliomas. The study opened for the accrual of patients on September 21, 1988, and was closed on June 30, 1991. The first 54 children in the study were treated with irradiation doses of 100 cGy given twice daily to a total dosage of 7200 cGy. The next 66 children were treated with a similar daily regimens to a total of 7800 cGy. Tumors were diagnosed by clinical and radiographic criteria. Decisions about the need for surgery were left to the discretion of the treating neurosurgeon; tissue diagnosis did not alter the therapy in patients with diffuse infiltrating tumors. We reviewed the neuroradiology and neurosurgery reports as well as the pathological specimens of children entered on the study. By magnetic resonance (MR) imaging criteria, tumors involved the majority of the brain stem in 76% of cases; only three patients had tumors localized to the midbrain or medulla. Operations were performed on 56 of 120 patients (47%). Cerebrospinal fluid shunts were inserted in 27 (23%) of the children; insertion of a shunt was the only operation in 11, and a shunt was inserted in conjunction with a tumor operation in 16. Tumor operations were performed in 45 (38%) of the patients; 24 had stereotactic biopsies, and 21 had craniotomies. Of the 21 patients who had craniotomies, only biopsies were performed in 11; partial tumor resections were performed in 5 patients and subtotal resection in 5.(ABSTRACT TRUNCATED AT 250 WORDS)

Treatment of acute lymphoblastic leukemia : 30 years' experience at St. Jude Children's Research Hospital

Abstract: Background Therapy for childhood lymphoblastic leukemia has evolved during the past three decades, but key questions about what are the least toxic, most effective forms of treatment remain unanswered because of the lack of comprehensive follow-up information. Methods To assess long-term outcome in the series of clinical trials conducted at St. Jude Hospital, we compared the results of treatment typical of four eras: exploratory combination chemotherapy (era 1, 1962 to 1966; 91 patients), regimens for the control of meningeal leukemia (era 2, 1967 to 1979; 825 patients), limited intensification of therapy (era 3, 1979 to 1983; 428 patients), and extended intensification of therapy (era 4, 1984 to 1988; 358 patients). (“Intensification” refers to strategies of systemic chemotherapy that are more aggressive than conventional ones.) The major end points were survival and event-free survival; we also calculated the relative risk of treatment failure and the rate of relapse or death after treatment ended (post...

Biologic importance of neuraminidase stalk length in influenza A virus.

Abstract: To investigate the biologic importance of the neuraminidase (NA) stalk of influenza A virus, we generated mutant viruses of A/WSN/33 (H1N1) with stalks of various lengths (0 to 52 amino acids), by using the recently developed reverse genetics system. These mutant viruses, including one that lacked the entire stalk, replicated in tissue culture to the level of the parent virus, whose NA stalk contains 24 amino acid residues. In eggs, however, the length of the stalk was correlated with the efficiency of virus replication: the longer the stalk, the better the replication. This finding indicates that the length of the NA stalk affects the host range of influenza A viruses. The NA stalkless mutant was highly attenuated in mice; none of the animals died even after intranasal inoculation of 10(6) PFU of the virus (the dose of the parent virus required to kill 50% of mice was 10(2.5) PFU). Moreover, the stalkless mutant replicated only in the respiratory organs, whereas the parent virus caused systemic infection in mice. Thus, attenuation of the virus with the deletion of the entire NA stalk raises the possibility of its use as live vaccines.