Mapping of the Pim-1 oncogene in mouse t-haplotypes and its use to define the relative map positions of the tcl loci t0(t6) and tw12 and the marker tf (tufted).

Pim-1 is an oncogene activated in mouse T-cell lymphomas induced by Moloney and AKR mink cell focus (MCF) viruses. Pim-1 was previously mapped to chromosome 17 by somatic cell hybrids, and subsequently to the region between the hemoglobin alpha-chain pseudogene 4 (Hba-4ps) and the alpha-crystalline gene (Crya-1) by Southern blot analysis of DNA obtained from panels of recombinant inbred strains. We have now mapped Pim-1 more accurately in t-haplotypes by analysis of recombinant t-chromosomes. The recombinants were derived from Tts6tf/t12 parents backcrossed to + tf/ + tf, and scored for recombination between the loci of T and tf. For simplicity all t-complex lethal genes properly named tcl-tx are shortened to tx. The Pim-1 gene was localized 0.6 cM proximal to the tw12 lethal gene, thus placing the Pim-1 gene 5.2 cM distal to the H-2 region in t-haplotypes. Once mapped, the Pim-1 gene was used as a marker for further genetic analysis of t-haplotypes. tw12 is so close to tf that even with a large number of recombinants it was not possible to determine whether it is proximal or distal to tf. Southern blot analysis of DNA from T-tf recombinants with a separation of tw12 and tf indicated that tw12 is proximal to tf. The mapping of two allelic t-lethals, t0 and t6 with respect to tw12 and tf has also been a problem.(ABSTRACT TRUNCATED AT 250 WORDS)     

Autocrine growth and progression of murine X-ray-induced T cell lymphomas.

Primary tumors of X-ray-induced murine T cell lymphomas comprise autocrine, growth factor-dependent cells. We have grown cell lines from primary X-ray-induced thymic lymphomas (PXTLs) under conditions which minimize the progression of the cells from factor dependence to factor independence. All (22) PXTL lines grown secrete a growth factor which supports their own growth and which we will call lymphoma growth factor LGF. LGF-dependent cells are non-tumorigenic or poorly tumorigenic, do not clone in soft agar, have no detectable rearrangements in the c-myc or Pim-1 region and possess near diploid or pseudodiploid karyotypes without evidence for trisomy of chromosomes nos. 15 or 17. PXTL-secreted LGF has no interleukin 1, 2, or 3 activity nor do LGF-secreting cells synthesize detectable IL-1, -2, or -3 mRNA. LGF contains no detectable interferon or GM-CSF activity in specific bioassays. Purified EGF, TGF beta, and interleukin preparations are inactive on LGF-dependent PXTL cells. Thus LGF appears to be a new growth factor that is required for the proliferation of non-progressed T lymphoma cells. Upon progression PXTL cells become growth factor independent, are highly tumorigenic in vivo, clone in soft agar, and assume a near triploid karyotype containing numerous chromosomal aberrations. Thus in X-ray-induced lymphomagenesis an autocrine, LGF-dependent phase precedes the progressed phase characterized by rearrangements in the myc and/or Pim-1 regions as well as by many chromosomal aberrations visible in the karyotype.     

Pim-1 ligand-bound structures reveal the mechanism of serine/threonine kinase inhibition by LY294002

Pim-1 is an oncogene-encoded serine/threonine kinase primarily expressed in hematopoietic and germ cell lines. Pim-1 kinase was originally identified in Maloney murine leukemia virus-induced T-cell lymphomas and is associated with multiple cellular functions such as proliferation, survival, differentiation, apoptosis, and tumorigenesis (Wang, Z., Bhattacharya, N., Weaver, M., Petersen, K., Meyer, M., Gapter, L., and Magnuson, N. S. (2001) J. Vet. Sci. 2, 167-179). The crystal structures of Pim-1 complexed with staurosporine and adenosine were determined. Although a typical two-domain serine/threonine protein kinase fold is observed, the inter-domain hinge region is unusual in both sequence and conformation; a two-residue insertion causes the hinge to bulge away from the ATP-binding pocket, and a proline residue in the hinge removes a conserved main chain hydrogen bond donor. Without this hydrogen bond, van der Waals interactions with the hinge serve to position the ligand. The hinge region of Pim-1 resembles that of phosphatidylinositol 3-kinase more closely than it does other protein kinases. Although the phosphatidylinositol 3-kinase inhibitor LY294002 also inhibits Pim-1, the structure of the LY294002.Pim-1 complex reveals a new binding mode that may be general for Ser/Thr kinases.     

Human homologs of two testes-expressed loci on mouse chromosome 17 map to opposite arms of chromosome 6

Our laboratory has recently cloned and characterized two testes-expressed loci--the Tcp-10 gene family cluster and the D17Si11 gene--that map to the proximal portion of mouse chromosome 17. Human homologs of both loci have been identified and cloned. Somatic cell hybrid lines have been used to map the human homolog of D17Si11 to the short arm of chromosome 6 (p11-p21.1) along with homologs of other genes from the (Pim-1)-(Pgk-2) region of the mouse chromosome. The human TCP 10 locus maps to the long arm of chromosome 6 (q21-qter) along with homologs of other genes from the mouse chromosome 17 region between the centromere and Pim-1. The mapping of large portions of the mouse t haplotype to unlinked regions on human chromosome 6 rules out the possibility that a t-haplotype-like chromosome could exist in humans.     

The Pim family of protein kinases: structure, functions and roles in hematopoietic malignancies

Phosphorylation is the universal regulatory mechanism in key physiological processes such as development, cell differentiation, proliferation, survival and malignant transformation. In this review we analyze serine/threonine protein kinases of the Pim (proviral integration of Moloney virus) family that have been initially discovered in experimental lymphomas. We provide data on gene structure, evolution, functions and substrates of Pim protein kinases. Focusing on Pim-1 as the major isoform, we analyze its role in the biology of hematopoietic malignancies. Pim-1 is a pro-proliferative and pro-survival protein kinase. It is constitutively active due to autophosphorylation, and its downstream partners positively regulate the cell cycle. Pim-1 cooperates with c-Myc oncoprotein in leukemogenesis; furthermore, Pim-1, like the Akt protein kinase, prevents cell death. Thus, Pim kinases are regarded as new therapeutic targets. Finally, we present an original test system f or screening of Pim inhibitors. In this test system the growth of a genetically engineered Escherichia coli strain in the presence of kanamycin is dependent on the phosphorylation of aminoglycoside-3' phosphotransferase VIII by Pim-1: pharmacological inhibition of this phosphorylation increases the bacterial cell lysis.     

Structural basis of constitutive activity and a unique nucleotide binding mode of human Pim-1 kinase

Pim-1 kinase is a member of a distinct class of serine/threonine kinases consisting of Pim-1, Pim-2, and Pim-3. Pim kinases are highly homologous to one another and share a unique consensus hinge region sequence, ER-PXPX, with its two proline residues separated by a non-conserved residue, but they (Pim kinases) have <30% sequence identity with other kinases. Pim-1 has been implicated in both cytokine-induced signal transduction and the development of lymphoid malignancies. We have determined the crystal structures of apo Pim-1 kinase and its AMP-PNP (5'-adenylyl-beta,gamma-imidodiphosphate) complex to 2.1-angstroms resolutions. The structures reveal the following. 1) The kinase adopts a constitutively active conformation, and extensive hydrophobic and hydrogen bond interactions between the activation loop and the catalytic loop might be the structural basis for maintaining such a conformation. 2) The hinge region has a novel architecture and hydrogen-bonding pattern, which not only expand the ATP pocket but also serve to establish unambiguously the alignment of the Pim-1 hinge region with that of other kinases. 3) The binding mode of AMP-PNP to Pim-1 kinase is unique and does not involve a critical hinge region hydrogen bond interaction. Analysis of the reported Pim-1 kinase-domain structures leads to a hypothesis as to how Pim kinase activity might be regulated in vivo.     

Comparative molecular genetic analysis of lymphomas from six inbred mouse strains.

Previous studies of 21 highly lymphomatous AKXD recombinant inbred mouse strains demonstrated correlations between lymphoma type, the somatic proviral DNA content of the lymphoma, and the frequency of virally induced rearrangements in eight common sites of viral integration (Myc, Pim-i, Pvt-1, Mlvi-1, Mlvi-2, Fis-1, Myb, and Evi-1). In this study we analyzed lymphomas from six inbred mouse strains, AKR/J, C58/J, HRS/J (hr/hr and hr/+), SJL/J, SEA/GnJ, and CWD/LeAgl, to determine whether these correlations are also evident in these strains. Mice of the AKR/J, C58/J, and HRS/J strains died exclusively of T-cell lymphomas. In contrast to earlier studies which showed a great disparity in the rate and incidence of lymphomas in HRS/J hr/hr and HRS/J hr/+ mice, we found a high incidence of T-cell lymphomas and the same mean age of onset of disease in both strains. SJL/J mice died primarily of pre-B-cell lymphomas, whereas CWD/LeAgl and SEA/GnJ mice died primarily of B-cell lymphomas. Somatically acquired mink cell focus-forming proviruses were detected only in T-cell lymphomas, whereas ecotropic proviruses were found in lymphomas from all hematopoietic cell lineages. No rearrangements were detected in the Fis-1, Mlvi-2, and Myb loci, whereas rearrangements were detected in the Mlvi-1, Myc, Pim-1, Pvt-1, and Evi-1 loci. Most rearrangements were found in T-cell lymphomas, and many were virally induced. These results are similar to those we obtained previously for lymphomas of 21 highly lymphomatous AKXD recombinant inbred mouse strains.     

Effect of Single Amino Acid Substitution Observed in Cancer on Pim-1 Kinase Thermodynamic Stability and Structure

Pim-1 kinase, a serine/threonine protein kinase encoded by the pim proto-oncogene, is involved in several signalling pathways such as the regulation of cell cycle progression and apoptosis. Many cancer types show high expression levels of Pim kinases and particularly Pim-1 has been linked to the initiation and progression of the malignant phenotype. In several cancer tissues somatic Pim-1 mutants have been identified. These natural variants are nonsynonymous single nucleotide polymorphisms, variations of a single nucleotide occurring in the coding region and leading to amino acid substitutions. In this study we investigated the effect of amino acid substitution on the structural stability and on the activity of Pim-1 kinase. We expressed and purified some of the mutants of Pim-1 kinase that are expressed in cancer tissues and reported in the single nucleotide polymorphisms database. The point mutations in the variants significantly affect the conformation of the native state of Pim-1. All the mutants, expressed as soluble recombinant proteins, show a decreased thermal and thermodynamic stability and a lower activation energy values for kinase activity. The decreased stability accompanied by an increased flexibility suggests that Pim-1 variants may be involved in a wider network of protein interactions. All mutants bound ATP and ATP mimetic inhibitors with comparable IC50 values suggesting that the studied Pim-1 kinase mutants can be efficiently targeted with inhibitors developed for the wild type protein.     

Sint1, a common integration site in SL3-3-induced T-cell lymphomas, harbors a putative proto-oncogene with homology to the septin gene family

The murine retrovirus SL3-3 is a potent inducer of T-cell lymphomas when inoculated into susceptible newborn mice. Previously, DNAs from twenty SL3-3-induced tumors were screened by PCR for provirus integration sites. Two out of 20 tumors demonstrated clonal provirus insertion into a common region. This region has now been isolated and characterized. The region, named SL3-3 integration site 1 (Sint1), maps to the distal end of mouse chromosome 11, corresponding to human chromosome 17q25, and may be identical to a mouse mammary tumor virus integration site in a T-cell lymphoma, Pad3. Two overlapping genomic lambda clones spanning about 35 kb were isolated and used as a starting point for a search for genes in the neighborhood of the virus integration sites. A genomic fragment was used as a hybridization probe to isolate a 3-kb cDNA clone, the expression of which was upregulated in one of two tumors harboring a provirus in Sint1. The cDNA clone is predicted to encode a protein which shows 97.0% identity to a human septin-like protein encoded by a gene which has been found as a fusion partner gene of MLL in an acute myeloid leukemia with a t(11;17)(q23;q25). Together these findings raise the possibility that a proto-oncogene belonging to the septin family, and located about 15 kb upstream of the provirus integration sites, is involved in murine leukemia virus-induced T-cell lymphomagenesis.     

Cellular DNA region involved in induction of thymic lymphomas (Mlvi-2) maps to mouse chromosome 15.

Two cellular DNA regions representing common domains for proviral DNA integration ( Mlvi -1 and Mlvi -2) have been identified in Moloney murine leukemia virus-induced rat thymic lymphomas. Cellular sequences which were free of repeated DNA derived from a clone that defines the Mlvi -2 integration domain (lambda Cl228 ) were found to be highly conserved in a variety of vertebrate species that we examined, including mice, hamsters, cats, and humans. In this study, we identified the chromosomal map location of the Mlvi -2 homologous sequences in mice by using hamster-mouse somatic cell hybrids. The results show that Mlvi -2 is present on mouse chromosome 15 but is unrelated to the c-myc and c-sis proto-oncogenes, which map on the same chromosome. Since aberrations on chromosome 15 have been observed reproducibly in mouse thymomas, our data suggest that Mlvi -2 may define a novel sequence involved in the induction or progression of murine thymic lymphomas.     

Sequence analysis of amphotropic and 10A1 murine leukemia viruses: close relationship to mink cell focus-inducing viruses.

Viral interference studies have demonstrated the existence of four distinct murine leukemia virus (MuLV) receptors on NIH 3T3 mouse cells. The four viral interference groups are ecotropic MuLV; mink cell focus inducing virus (MCF); amphotropic MuLV; and 10A1, a recombinant derivative of amphotropic MuLV that uses a unique receptor but also retains affinity for the amphotropic MuLV receptor. We report here that 10A1 infects rat and hamster cells, unlike its amphotropic parent. We isolated an infectious molecular clone of 10A1 and present here the sequences of the env genes and enhancer regions of amphotropic MuLV and 10A1. The deduced amino acid sequences of amphotropic MuLV and 10A1 gp70su are remarkably similar to those of MCF and xenotropic MuLV (for which mouse cells lack receptors), with 64% amino acids identical in the four groups. We generated a consensus from these comparisons. Further, the differences are largely localized to a few discrete regions: (i) amphotropic MuLV has two short insertions relative to MCF, at residues 87 to 92 and 163 to 169, and (ii) amphotropic MuLV and MCF are totally different in a hypervariable region, which is greater than 30% proline, at residues approximately 253 to 304. 10A1 closely resembles amphotropic MuLV in its N terminus but contains an MCF-type hypervariable region. These results suggest the possibility that receptor specificity is localized in these short variable regions and further that the unique receptor specificity of 10A1 is due to the novel combination of amphotropic MuLV and MCF sequences rather than to the presence of any novel sequences. The Env proteins of ecotropic MuLV are far more distantly related to those of the other four groups than the latter are to each other. We also found that the enhancer regions of amphotropic MuLV and 10A1 are nearly identical, although 10A1 is far more leukemogenic than amphotropic MuLV.     

Elevated levels of oncogenic protein kinase Pim-1 induce the p53 pathway in cultured cells and correlate with increased Mdm2 in mantle cell lymphoma

Mutation of the p53 gene is a common event during tumor pathogenesis. Other mechanisms, such as mdm2 amplification, provide alternative routes through which dysfunction of the p53 pathway is promoted. Here, we address the hypothesis that elevated expression of pim oncogenes might suppress p53 by regulating Mdm2. At a physiological level, we show that endogenous Pim-1 and Pim-2 interact with endogenous Mdm2. Additionally, the Pim kinases phosphorylate Mdm2 in vitro and in cultured cells at Ser(166) and Ser(186), two previously identified targets of other signaling pathways, including Akt. Surprisingly, at high levels of Pim expression, as would occur in tumors, active, but not inactive, Pim-1 or Pim-2 blocks the degradation of both p53 and Mdm2 in a manner that is independent of Mdm2 phosphorylation, leading to increased p53 levels and, proportionately, p53-dependent transactivation. Additionally, Pim-1 induces endogenous ARF, p53, Mdm2, and p21 in primary murine embryo fibroblasts and stimulates senescence-associated beta-galactosidase levels, consistent with the induction of senescence. Immunohistochemical analysis of a cohort of 33 human mantle cell lymphomas shows that elevated expression of Pim-1 occurs in 42% of cases, with elevated Pim-2 occurring in 9% of cases, all of which also express Pim-1. Notably, elevated Pim-1 correlates with elevated Mdm2 in MCL with a p value of 0.003. Taken together, our data are consistent with the idea that Pim normally interacts with the p53 pathway but, when expressed at pathological levels, behaves as a classic dominant oncogene that stimulates a protective response through induction of the p53 pathway.     

Pim-1 kinase stability is regulated by heat shock proteins and the ubiquitin-proteasome pathway

Elevated expression of the serine/threonine kinase Pim-1 increases the incidence of lymphomas in Pim-1 transgenic mice and has also been found to occur in some human cancers. Pim-1 acts as a cell survival factor and may prevent apoptosis in malignant cells. It was therefore of interest to understand to what extent maintenance and degradation of Pim-1 protein is affected by heat shock proteins (Hsp) and the ubiquitin-proteasome pathway in K562 and BV173 human leukemic cells. The half-life of Pim-1 protein in these cells was found to increase from 1.7 to 3.1 hours when induced by heat shock or by treating the cells with the proteasome inhibitor PS-341 (bortezomib). The Hsp90 inhibitor geldanamycin prevented the stabilization of Pim-1 by heat shock. Using immunoprecipitation, it was determined that Pim-1 is targeted for degradation by ubiquitin and that Hsp70 is associated with Pim-1 under these circumstances. Conversely, Hsp90 was found to protect Pim-1 from proteasomal degradation. A luminescence-based kinase assay showed that Pim-1 kinase bound to Hsp70 or Hsp90 remains active, emphasizing the importance of its overall cellular levels. This study shows how Pim-1 levels can be modulated in cells through degradation and stabilization.     

Sequential events of apoptosis involving docetaxel, a microtubule-interfering agent: A cytometric study

Background  

Pim-1, 2 and 3 are a group of enzymes related to the calcium calmodulin family of protein kinases. Over-expression of Pim-1 and Pim-2 in mice promotes the development of lymphomas, and up-regulation of Pim expression has been observed in several human cancers.     

The serine/threonine kinase Pim-1

The human pim-1 gene encodes a serine/threonine kinase, which belongs to the group of calcium/calmodulin-regulated kinases (CAMK). It contains a characteristic kinase domain, a so-called ATP anchor and an active site. In mouse and human, two Pim-1 proteins are produced from the same gene by using an alternative upstream CUG initiation codon, a 44 kD and another, shorter 34 kD form that both contain the kinase domain. Expression of Pim-1 is widespread and ranges from the hematopoietic and lymphoid system to prostate, testis and oral epithelial cells. Two other proteins with significant sequence similarities exist, Pim-2 and Pim-3; both are also serine/threonine kinases and have largely overlapping functions. Pim-1 is able to phosphorylate different targets, most of which are involved in cell cycle progression or apoptosis. Pim-1 expression can be induced by several external stimuli in particular by a number of cytokines relevant in the immune system, which led to the labeling of Pim-1 as a "booster" for the immune response.