Neoplastic transformation by the gep oncogene, Galpha12, involves signaling by STAT3.

Galpha(12), the alpha-subunit of G12, which has been referred to as the gep oncogene, stimulates mitogenic pathways in different cell types and readily induces neoplastic transformation of fibroblast cell lines. Recently, we have shown that the oncogenic pathway activated by Galpha(12) involves the receptor tyrosine kinase platelet derived growth factor receptor-alpha (PDGFRalpha) and JAK3. In the present study, we demonstrate that the GTPase-deficient activated mutant of Galpha(12) activates signal transducer and activator of transcription 3 (STAT3) via PDGFRalpha as well as JAK3. Here we show that Galpha(12) stimulates the phosphorylation of STAT3 at both Tyrosine-705 and Serine-727 residues. Studies to delineate the mechanism by which Galpha(12) stimulates STAT3 have indicated that the Tyrosine-705-phosphorylation of STAT3 involves the tyrosine kinases, Janus Kinase-3 as well as Src kinase, whereas the Serine-727 phosphorylation of STAT3 occurs via the receptor tyrosine kinase, PDGFRalpha and phosphatidylinositol 3-OH kinase pathway. Our results also indicate that the coexpression of the dominant negative, DNA binding mutant of STAT3 (STAT3DB) inhibits the foci formation as well as anchorage-independent growth of Galpha(12)QL-transfectants, thereby establishing the critical role of STAT3 in Galpha(12)QL-mediated neoplastic cell growth. The results presented here demonstrate, for the first time, the ability of Galpha(12) to recruit multiple receptor-, nonreceptor-, and Ser/Thr kinases to stimulate STAT3-signaling to promote neoplastic transformation.

Src: regulation, role in human carcinogenesis and pharmacological inhibitors.

The cellular signaling machinery is a complex network of cross-talking proteins that enables dynamic communication between upstream causal factors and downstream effectors. Non-receptor tyrosine kinases, including Src, are the intermediates of signal transfer, controlling pathways as diverse as cell growth, death, differentiation, migration, and genome maintenance. When expressed as viral genes these proteins are potent carcinogens. Furthermore, analogous genetic alterations are observed, albeit not frequently, in human tumors. In a variety of tumors including those derived from the colon and breast, Src is either over expressed or constitutively active in a large percentage of patients. Increased expression or activity of Src correlates with the stage and metastatic potential of some neoplasia. The detailed knowledge of Src activation facilitates rational design of drugs that potentially interfere with either binding of ATP or substrate peptides. Several existing inhibitors are available as lead compounds for further development of Src inhibitors.

Cisplatin-resistance involves the defective processing of MEKK1 in human ovarian adenocarcinoma 2008/C13 cells.

Cisplatin has been widely used as a chemotherapeutic agent to treat different types of tumors. However, its use is limited by the ability of the tumor cells to develop cisplatin-resistance. The molecular lesion that produces cisplatin-resistance is poorly understood. In this report, we show that cisplatin activates a robust apoptotic pathway involving the activation of JNK and p38MAPK whereas it fails to elicit such a response in cisplatin-resistant 2008/C13 cells. Analysis of the defective apoptotic pathway in 2008/C13 cells indicates that these cells are deficient in the proteolytic activation of MEKK1 by caspase-3. The blunted activity of caspase-3 appears to be closely related to the increased levels of the anti-apoptotic protein Bcl-xL seen in the resistant cells. These studies, for the first time, demonstrate that inadequate caspase-3 processing and MEKK1 activation can lead to a drug-resistant phenotype.

Tyrosine phosphorylation of C-Cbl facilitates adhesion and spreading while suppressing anchorage-independent growth of V-Abl-transformed NIH3T3 fibroblasts.

The protooncogenic protein c-Cbl becomes tyrosine phosphorylated in normal cells in response to a variety of external stimuli, as well as in cells transformed by oncogenic protein tyrosine kinases. Tyrosine phosphorylation of c-Cbl upregulates its binding to multiple crucial signaling molecules. However, the biological consequences of c-Cbl-mediated signaling are insufficiently understood. To analyse the biological functions of c-Cbl, we overexpressed wild-type c-Cbl and its tyrosine phosphorylation-defective mutant form in v-Abl-transformed NIH3T3 fibroblasts. In this system, wild-type c-Cbl facilitated adhesion and spreading of v-Abl-transformed fibroblasts on the extracellular matrix, while reducing anchorage independence of these cells, as measured by their colony-forming efficiency in soft agar. Therefore, overexpression of wild-type c-Cbl exhibits an overall transformation-suppressing effect. By contrast, overexpression of a tyrosine phosphorylation-defective form of c-Cbl increases neither adhesion nor anchorage dependence of v-Abl-transformed fibroblasts. Analysis of the role of individual tyrosine phosphorylation sites of c-Cbl in these phenomena indicates that both phosphatidylinositol-3' kinase and the CrkL adaptor protein may be involved in the observed effects of c-Cbl. To summarize, the results presented in this report indicate that c-Cbl is involved in regulation of cell adhesion and cytoskeletal rearrangements, and that these effects of c-Cbl are dependent on its tyrosine phosphorylation.

Molecular cloning of platelet factor XI, an alternative splicing product of the plasma factor XI gene.

Platelet factor XI is associated with the platelet plasma membrane and has an apparent Mr (220,000 nonreduced, 55,000 reduced) different from that of plasma factor XI. However, the site of synthesis and the nature of platelet factor XI are not known. Using reverse transcriptase polymerase chain reaction, 12 out of 13 exons (all except exon V) coding for mature plasma factor XI were amplified from human platelet mRNA. The sequence of each of these exons was identical to that of plasma factor XI. In situ amplification and hybridization of factor XI mRNA was positive for exon III and negative for exon V in platelets and negative for both exons in other blood cells. By Northern hybridization, a factor XI mRNA transcript of approximately 1.9 kilobases was detected in megakaryocytic cells, and one of approximately 2.1 kilobases was detected in liver cells. Factor XI cDNA was cloned from a megakaryocyte library and sequenced. Exon V was absent, and the splicing of exon IV to exon VI maintained the open reading frame without alteration of the amino acid sequence except for the deletion of amino acids Ala91-Arg144 within the amino-terminal portion of the Apple 2 domain. Thus, platelet factor XI is an alternative splicing product of the factor XI gene, localized to platelets and megakaryocytes but absent from other blood cells.

c-ABL tyrosine kinase activity is regulated by association with a novel SH3-domain-binding protein.

The c-ABL tyrosine kinase is activated following either the loss or mutation of its Src homology domain 3 (SH3), resulting in both increased autophosphorylation and phosphorylation of cellular substrates and cellular transformation. This suggests that the SH3 domain negatively regulates c-ABL kinase activity. For several reasons this regulation is thought to involve a cellular protein that binds to the SH3 domain. Hyperexpression of c-ABL results in an activation of its kinase, the kinase activity of purified c-ABL protein in the absence of cellular proteins is independent of either the presence or absence of a SH3 domain, and point mutations and deletions within the SH3 domain are sufficient to activate c-ABL transforming ability. To identify proteins that interact with the c-ABL SH3 domain, we screened a cDNA library by the yeast two-hybrid system, using the c-ABL SH3SH2 domains as bait. We identified a novel protein, AAP1 (ABL-associated protein 1), that associates with these c-ABL domains and fails to bind to the SH3 domain in the activated oncoprotein BCRABL. Kinase experiments demonstrated that in the presence of AAP1, the ability of c-ABL to phosphorylate either glutathione S-transferase-CRK or enolase was inhibited. In contrast, AAP1 had little effect on the phosphorylation of glutathione S-transferase-CRK by the activated ABL oncoproteins v-ABL and BCRABL. We conclude that AAP1 inhibits c-ABL tyrosine kinase activity but has little effect on the tyrosine kinase activities of oncogenic BCRABL or v-ABL protein and propose that AAP1 functions as a trans regulator of c-ABL kinase. Our data also indicate that loss of susceptibility to AAP1 regulation correlates with oncogenicity of the activated forms of c-ABL.

Multi-unit anti-BCR-ABL ribozyme therapy in chronic myelogenous leukemia.

In this review, we summarize and update our data on the development of a multi-unit anti-BCR/ABL ribozyme. In vitro studies comparing several anti-BCR/ABL ribozymes demonstrated that a triple-unit ribozyme is the most efficient. Detailed kinetic analysis revealed this ribozyme to have a lower Kcat, most likely due to non homologous bases at restriction enzyme sites used in ribozyme construction. Delivery of this ribozyme to a BCR/ABL transformed cell line by a novel vehicle targeting the folate receptor resulted in a 3 log reduction in BCR/ABL mRNA when analyzed by RT-PCR. This delivery strategy reversed the IL-3 independence of this cell line. Retroviral vectors containing genes coding for the multi-unit ribozyme have been constructed and their use to effect BCR/ABL transformed cell biology is discussed.

Activated abl oncogenes and apoptosis: differing responses of transformed myeloid progenitor cell lines.

Activation of the c-abl protooncogene occurs during the generation of both the Abelson murine leukemia virus and the bcrabl fusion gene. To further dissect the biological properties of these proteins, we studied their effect on apoptosis. Using dimethyl sulfoxide (DMSO) to induce apoptosis in the murine myeloid progenitor cell line 32Dcl3, we examined the effect of expression of both v-abl and bcrabl transgenes on apoptosis. v-abl expressing 32Dcl3 cells are sensitive to apoptosis induction, similar to parental 32Dcl3 cells. In contrast, bcrabl expression 32Dcl3 cells are protected from the apoptotic stimulus resulting from DMSO exposure. Analyzing the expression patterns for Bcl-2 and Bax, two proteins known to modulate the apoptotic response, we found a downregulation of Bcl-2 and enhanced expression of Bax in 32Dcl3 cells. In 32Dcl3/v-Abl cells, Bcl-2 expression remained constant while Bax was upregulated, whereas in 32Dcl3 cells expressing bcrabl, there was continuous expression of Bcl-2 at a level greater than observed in v-abl transformed cells. Taken together, our data demonstrate that although both activated abl gene products promote overlapping effects of some biological responses (i.e., factor-independent proliferation) they diverge in their effect on apoptotic signaling pathways.

Scintigraphic imaging of oncogenes with antisense probes: does it make sense?

Based on the specificity of the Watson-Crick base pairing formation, antisense deoxyoligonucleotides have been used to inhibit the expression of oncogenes in various cancer cells. Activation of an oncogene by means of amplification leads to an increased, detectable amount of the mRNA transcript in the cytoplasm. The aim of this study was to demonstrate that cells which are expressing a particular mRNA transcript do preferentially and specifically retain the antisense probe targeting that mRNA. Using a mouse plasmacytoma cell line (MOPC315) which produces high levels of IgA heavy chain mRNA, a control mouse pre B cell line (7OZ/3B), a human mammary cell line (MCF7) which expresses the erbB2 or neu oncogene, MOPC315 cells as neu-negative controls, and antisense DNA oligonucleotides complementary to the 5' region of the mRNAs and the sense sequence, we have shown that there is a preferential, specific retention of the IgA and neu antisense sequence in MOPC315 and MCF7 cells, respectively. We have further demonstrated that this retention is time and concentration dependent with a maximum at 24 h. We conclude that cancer cells which express a particular oncogene are suitable targets for radiolabeled antisense deoxyoligonucleotides directed toward the oncogene transcript. This work and recent developments in the antisense field lead to the expectation of a new class of radiopharmaceuticals with unique specificity.

Multi-unit ribozyme-mediated cleavage of bcr-abl mRNA in myeloid leukemias.

Chronic myelogenous leukemia is characterized by the Philadelphia chromosome, which at the molecular level results from the fusion of the bcr gene on chromosome 22 and the abl gene on chromosome 9. The bcr-abl fusion gene encodes a novel tyrosine kinase with transforming activity. In this study, we have synthesized a multi-unti ribozyme that targets bcr-abl mRNA. In vitro ribozyme cleavage reactions show increased cleavage efficiency of this multi-unit ribozyme compared with single or double ribozymes. The multiunit ribozyme was then transfected into murine myeloblasts transformed with the bcr-abl gene (32D cells). Ribozyme transfection was accomplished either by liposomes or using follic acid-polylysine as a carrier. Multi-unit ribozyme transfection reduced the level of bcr-abl mRNA 3 logs when transfected via folate receptor-mediated uptake into transformed 32D cells. These results suggest that a multi-unit ribozyme could be an effective therapeutic agent for the treatment of Philadelphia chromosome-positive chronic myelogenous leukemia.

Activated mutant of G alpha 13 induces Egr-1, c-fos, and transformation in NIH 3T3 cells.

Expression of the constitutively activated mutant alpha-subunit of the heterotrimeric G protein G alpha 13 (alpha 13Q226L) leads to the transformation of NIH-3T3 cells. An analysis of the mitogenic pathway mediated by alpha 13Q226L indicated that the expression of the primary response genes, early growth response gene-1 (Egr-1, a nuclear transcription factor with zinc-finger motif) and c-fos (a leucine zipper transcription factor as well as a protooncogene) are constitutively activated in alpha 13Q226L-transformants. While ras-transformed cells did not express Egr-1, cells transformed by the GTPase deficient mutant alpha-subunit of G alpha 12 (alpha 12Q229L) exhibited a "weak" expression, suggesting that the induction of Egr-1 and c-fos is intrinsic to G alpha 13 signaling pathway and not a consequence of the transformed phenotype. Taken together, these results provide the first evidence that the G alpha 13 signaling pathway involves the activation of specific transcription factors and defines the expression of these nuclear transcription factors as a possible molecular mechanism in alpha 13Q226L-mediated cell proliferation and transformation.

Structural alterations in the carboxyl-terminal domain of the BCRABL gene product activate its fibroblastic transforming potential.

Activation of the c-abl protooncogene occurs in Abelson murine leukemia virus, Hardy-Zuckerman-2 feline sarcoma virus, and during the chromosomal translocation that generates the BCRABL fusion gene. The three genes exhibit varying degrees of transforming activity; the two viral genes transform NIH-3T3 cells in vitro, whereas the BCRABL gene is incapable of transforming these cells. To determine whether genetic alterations can enhance the transforming potential of the BCRABL gene, we employed genetic selection techniques which led to the isolation of a mutant form of the BCRABL gene with high levels of fibroblastic transforming activity. Molecular analysis of this clone shows that it suffered a deletion of 3' ABL sequences and their replacement with a cellular sequence of unknown origin, termed X. This tripartite gene is capable of inducing 35 foci/10 ng of DNA. Deletion of 3' ABL sequences analogous to that seen in the activated BCRABL protein without the addition of X yields 5 foci/100 ng of DNA. These results suggest that carboxyl-terminal truncations unmask the fibroblastic transforming activity of the BCRABL gene product and the addition of X sequences dramatically enhances this transforming potential, indicating a dominant contribution by the X reading frame.

Ribozyme-mediated cleavage of the BCRABL oncogene transcript: in vitro cleavage of RNA and in vivo loss of P210 protein-kinase activity.

The t(9,22) chromosomal translocation generating the Philadelphia chromosome and the BCRABL oncogene has been shown both cytogenetically and molecularly to be the etiologic event in chronic myelogenous leukemia (CML). We have designed a ribozyme to cleave the BCRABL mRNA by targeting a GUU triplet adjacent to the junction of the c-BCR and c-ABL fused genes. This ribozyme efficiently cleaved BCRABL RNA transcripts as demonstrated by in vitro cleavage reactions. To determine the effect of constitutive expression of the ribozyme on the elimination of the BCRABL gene product, the ribozyme cDNA sequence was inserted into different retroviral expression vectors. Introduction of the recombinant retroviruses into the CML blast crisis cell-line K562, resulted in the elimination of the P210 protein-kinase activity in several single cell clones infected with the ribozyme expression cassette. Therefore BCR-ABL specific ribozymes may provide a potential genetic therapy for CML.

Activation of murine c-abl protooncogene: effect of a point mutation on oncogenic activation.

Activation of the c-abl protooncogene occurs in Abelson murine leukemia virus, in Hardy-Zuckerman 2 feline sarcoma virus, and during the chromosomal translocations that generate BCR-ABL gene fusion products. To study the molecular mechanism involved in the c-abl activation, we have created a series of modifications in murine c-abl and assayed these constructs for oncogenic activity using the NIH 3T3 cell transformation assay. Our results show that amino-terminal deletions are sufficient for oncogenic activation of c-abl and high levels of oncogenic activities were generated by a deletion of 114 codons from the 5' end that deleted the SH3 region. A deletion of 53 codons from the 5' end (inclusive of deletions seen in Hardy-Zuckerman 2 feline sarcoma virus and BCR-ABL gene products) that retains the SH3 region of c-abl resulted in the generation of low levels of transforming activity. This transforming potential was substantially increased with the introduction of a G----A point mutation in codon 832 that is present in v-abl. The point mutation was found to affect the secondary structure and the tyrosine kinase activity of the mutant gene products.

Transformation of NIH3T3 cells by A-MuLV proviral DNA: effect of plasmid linearization and carrier DNA on transformation efficiency.

We have optimized the conditions for efficient NIH3T3 focus formation by calcium phosphate transfection of proviral Abelson-murine leukemia virus (A-MuLV) plasmid DNA. Linearized pA-MuLV, P120 or P160 strains, when transfected with calf thymus carrier DNA, will produce 40-50 foci/100 ng pA-MuLV without co-transfecting Moloney-murine leukemia virus (Mo-MuLV) plasmid DNA.

Deregulation and overexpression of c-fos proto-oncogene in rat renal cell-lines and primary tumors induced by dimethylnitrosamine.

Rat renal mesenchymal tumors induced by the chemical carcinogen N-dimethylnitrosamine (DMN) and cell-lines derived from kidneys of rats after DMN treatment were found to express abnormal steady state levels of c-fos RNA. This overexpression was not found to arise by gene amplification or rearrangement, but the c-fos gene appeared to be deregulated resulting in increased transcription. The consistent nature of this observation suggests a function for c-fos gene overexpression in tumorigenesis of the rat kidney by DMN.

Nucleotide sequence of testis-derived c-abl cDNAs: implications for testis-specific transcription and abl oncogene activation.

The c-abl gene codes for a protein-tyrosine kinase and is expressed in most examined murine cell types as two distinct mRNA species of 5.5 kilobases (kb) and 6.5 kb. In mouse testis, an additional species of 4.0 kb is expressed in very high levels. To study the interrelationship between various c-abl transcripts and to compare their sequence with the v-abl transcript, we prepared c-abl-specific cDNA clones from mouse testis and determined the complete nucleotide sequence of the 4.0-kb cDNA that appears to be the reverse transcript of the testis-specific mRNA. In addition, we have determined the 3' sequence of an additional clone derived from the larger mRNA species that is expressed in somatic as well as germ-line cells. These cDNA sequences have been compared with the v-abl sequences to understand the mechanism of activation of this oncogene. The results demonstrate that (i) testis-specific c-abl mRNAs arise as a result of 3' truncation, and (ii) the v-abl gene has arisen from its cellular homologue as a result of an extensive deletional/mutational process.

Cloning and characterization of a rat-specific repetitive DNA sequence.

A 2.1-kb EcoRI fragment of rat DNA has been cloned and sequenced. This fragment contained a repetitive element which was highly specific for rat DNA and widely dispersed throughout the rat genome. The repetitive element is homologous to a sequence found in the 3' end of the rat LINE family. Because of its high degree of species specificity and its heterodisperse distribution, this sequence provided a useful marker for rat DNA in DNA transfection experiments into mouse host cells.