A rapid method for the preparation of yeast lysates that facilitates the immunodetection of proteins generated by the yeast two-hybrid system.
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Blotting, Western↗
Biomedical subjects
E V Prochownik
Publications and source records attributed to E V Prochownik.
Mutation of the MXI1 gene in prostate cancer.
The Mxi1 protein negatively regulates Myc oncoprotein activity and thus potentially serves a tumour suppressor function. MXI1 maps to chromosome 10q24-q25, a region that is deleted in some cases of prostate cancer. We have detected mutations in the retained MXI1 alleles in four primary prostate tumours with 10q24-q25 deletions. Two tumours contained inactivating mutations, whereas two others contained the identical missense mutation. Fluorescence in situ hybridization also demonstrated loss of one MXI1 allele in an additional tumour lacking chromosome 10 abnormalities. MXI1 thus displays allelic loss and mutation in some cases of prostate cancer that may contribute to the pathogenesis or neoplastic evolution of this common malignancy.
Alleles↗
Proximal promoter region of the junB gene mediates attenuation of serum inducibility in Src-transformed cells.
Transcription of the junB gene is rapidly and transiently induced following stimulation of susceptible cells by growth factors in serum. Our previous studies demonstrated that serum inducibility of junB and other immediate-early genes is markedly attenuated in fibroblasts chronically transformed by the viral Src oncoprotein. Moreover, attenuation of junB induction occurs at the transcriptional level. To characterize further the molecular mechanisms of this attenuation, various full-length and recombinant junB constructs were transfected into normal and viral Src-transformed rat fibroblasts. A stable transfection system was used to faithfully reproduce regulation of the junB gene. Analyses of pooled populations of stably transfected cells demonstrate that sequences between -89 and +32 (relative to the transcriptional start site) are sufficient to confer both serum inducibility of the junB gene in normal cells and its attenuation in viral Src-transformed cells. By contrast, attenuation of c-fos serum inducibility by Src transformation involves element(s) distinct from regulatory elements identified previously in the c-fos promoter. These results identify a proximal promoter region of the junB gene that is involved in a novel negative regulation of its transcription.
Animals↗
Assignment of the human MAD and MXI1 genes to chromosomes 2p12-p13 and 10q24-q25.
MAD and MXI1, two recently described members of the basic helix-loop-helix (bHLH) gene family, encode proteins that dimerize with and modulate the DNA binding of max. In turn, mad-max or mxi1-max heterodimers or max homodimers can compete for DNA binding sites with dimers formed between max and myc oncoproteins and antagonize the transcriptional activities of this latter class of proteins. Using a combination of somatic cell mapping and fluorescence in situ hybridization techniques, we have determined the chromosomal locations of the MAD and MXI1 genes. The MAD gene maps to chromosome 2p12-p13, a region involved in translocations and deletions in acute and chronic lymphocytic leukemias as well as non-lymphocytic leukemias and Hodgkin disease. The MXI1 gene localizes to chromosome 10q24-q25, a region involved in translocations and deletions in acute and chronic lymphocytic leukemias and prostatic carcinomas. The availability of genomic clones of MAD and MXI1 will permit an assessment of their involvement in these diseases at the molecular level.
Animals↗
Increased cell-substrate adhesion accompanies conditional reversion to the normal phenotype in ras-oncogene-transformed NIH-3T3 cells.
We recently reported (1991, Mol. Cell Biol. 11, 3699-3710) that depletion of c-myc protein by myc antisense sequences in ras-transformed NIH-3T3 cells reverses several of the malignant characteristics of these cells. These include transformed morphology, growth in soft agar, and ability to form tumors in athymic mice. In the present study we examined the same cells for in vitro adhesive behavior. Cells depleted of c-myc protein by antisense transfection showed no change in attachment to fibronectin-coated dishes as compared to ras-transformed NIH-3T3 cells but had greatly increased resistance to trypsin/EDTA-mediated release from the substratum after attachment. In concomitant studies, the cells were examined for fibronectin biosynthesis and cell surface fibronectin. There was no overall change in fibronectin biosynthesis in the c-myc antisense transfected cells as compared to the ras-transformed NIH-3T3. However, immunofluorescence staining revealed increased amount of surface fibronectin associated with the antisense c-myc-expressing transfectants. Taken together, these data indicate that the conditional reacquisition of the nonmalignant phenotype in ras-transformed NIH-3T3 cells by selected depletion of c-myc protein is associated with an increase in cell-substrate adhesion. This, in turn, is associated with an increase in surface fibronectin.
3T3 Cells↗
Rhabdomyosarcomas do not contain mutations in the DNA binding domains of myogenic transcription factors.
Skeletal myogenesis is regulated by a group of transcription factors (MyoD, myogenin, myf5, and myf6) that are "basic helix-loop-helix" proteins that bind to the promoters of muscle-specific genes and promote their expression. We have previously shown that after a mutation of Leu122 to Arg the DNA binding basic domain of MyoD confers c-myc-like functional characteristics to the protein. In this study we used single-strand conformation polymorphism analysis to determine whether such mutations occur naturally in rhabdomyosarcomas. We have found that the basic domains of all the myogenic factors remain unaltered in rhabdomyosarcomas. Selection against such mutations may be the result of functional redundancy of these myogenic transcription factors.
Amino Acid Sequence↗
Differential patterns of DNA binding by myc and max proteins.
c-myc, N-myc, and L-myc genes are subject to highly variable degrees of tissue-specific regulation. Their aberrant expression has also been implicated in the pathogenesis of a variety of malignant tumors. The recently identified max protein dimerizes with c-myc to promote its sequence-specific DNA binding. max exists in two forms (long and short) that differ by virtue of a 9-amino acid insertion/deletion at the N terminus. We tested recombinant myc and max proteins for binding to six oligonucleotides containing related c-myc sites. Each myc protein, alone and in association with max proteins, manifested a unique pattern of DNA binding. Phosphorylation of both max proteins was observed when they were incubated in a rabbit reticulocyte lysate. This strongly affected DNA binding by max(long) but not by max(short). Our results point to the existence of specific DNA binding preferences for each of the myc proteins. The 9-amino acid segment that distinguishes max(long) from max(short) appears to serve a regulatory function that provides additional control over DNA sequence recognition.
Base Sequence↗
Attenuation of serum inducibility of immediate early genes by oncoproteins in tyrosine kinase signaling pathways.
Immediate early genes involved in controlling cell proliferation are rapidly and transiently induced following stimulation of susceptible cells with serum. To study how oncoproteins regulate immediate early genes, we examined serum inducibility of these genes in cells transformed by various oncoproteins. We found that induction of the immediate early gene, c-fos, by serum stimulation was markedly attenuated in four independent cell lines stably transformed by the v-Src tyrosine kinase. Cells chronically transformed by other oncoproteins implicated in tyrosine kinase signaling pathways, including v-Sis, v-Ras, and v-Raf, showed the same pattern of attenuation. In contrast, serum inducibility of c-fos was not attenuated in cells transformed by simian virus 40, which is thought to transform cells through a different pathway. Cell cycle analyses showed that proliferation of these transformed cell lines could be arrested effectively in 0.1% serum, demonstrating that the attenuation was not simply due to continuous cycling of transformed cells after serum deprivation. Moreover, serum inducibility of other immediate early genes, including c-jun, junB, egr-1, and NGFI-B, also was strikingly attenuated by these same oncoproteins. Nuclear run-on transcription assays established that this attenuation of serum inducibility occurred at the transcriptional level. Finally, flow cytometric analysis demonstrated that serum-starved v-Src-transformed cells were viable and able to progress into S phase of the cell cycle after serum stimulation, even though the induction of immediate early genes was greatly attenuated in these cells. Our results suggest that activation of immediate early genes is repressed by chronic stimulation of tyrosine kinase signaling pathways in transformed cells.
Animals↗
A point mutation in the MyoD basic domain imparts c-Myc-like properties.
MyoD and c-Myc, members of the large "basic-helix-loop-helix" family of proteins, regulate diverse aspects of both normal and neoplastic growth and specific gene regulation. These two proteins differ at 9 of the 14 amino acids that comprise the basic domains necessary for DNA binding and transcriptional control. Individual amino acids in the MyoD basic domain were mutated to those found at the analogous positions in c-Myc. Four classes of mutants were obtained: (i) those with no effects on MyoD-site binding or activation of MyoD-responsive genes, (ii) those with no effect on MyoD-site binding but with a loss of activation potential, (iii) those with a loss of both DNA binding and activation potential, and (iv) one mutant (mut 9, Leu122----Arg) that left MyoD-site binding unaffected but imparted a new c-Myc-site binding capability. mut 9 competed with wild-type protein for the activation of MyoD-responsive reporter genes but could, like c-Myc, also suppress the adenovirus major-late promoter, which contains a c-Myc binding site. Our studies thus identify specific amino acid residues in the MyoD basic domain that are important for its activity as a DNA-binding transcriptional activator. Most significantly, our results with mut 9 indicate that Leu122 of MyoD is a critical determinant of specific DNA binding and that mutation at this residue can alter this specificity.
Amino Acid Sequence↗
Inhibition of c-jun causes reversible proliferative arrest and withdrawal from the cell cycle.
We studied the effect of c-jun depletion in Friend murine erythroleukemia (F-MEL) cells stably transfected with a plasmid that allowed for the glucocorticoid-mediated conditional expression of c-jun antisense sequences. The c-jun cDNA used for the construction of the vector was modified so as to prevent the nonspecific targeting of junB and junD transcripts. High level and rapid induction of c-jun antisense transcripts was achieved with as little as 10(-8) mol/L dexamethasone (DEX) and resulted in a 80% to 90% reduction in c-jun protein levels. The continuous exposure of the transfected cells to DEX inhibited growth by greater than 85% over a 5-day period, whereas DEX had no effect on the growth rate of control F-MEL cells. This proliferative block was associated with a reversible accumulation of cells with a 2n DNA content. When these cells were recultured in the absence of DEX, c-jun protein rapidly reappeared and the immediate early response genes egr-1, junB, and c-myc were transiently expressed. Thus, inhibition of c-jun protein causes logarithmically growing cells to leave the cell cycle and to enter a state closely resembling, if not identical to, G0. These results underscore the importance of c-jun in maintaining cellular proliferation and provide additional evidence for the participation of proto-oncogenes in cell cycle control.
Animals↗
Regulation of the junB gene by v-src.
The proteins encoded by cellular and viral src genes are believed to be involved in the transmission of mitogenic signals, the nuclear recipients of which are largely unknown. In this work, we report that four different v-src-transformed cell lines from three different species possess elevated levels of junB transcripts. Transient expression of junB promoter-chloramphenicol acetyltransferase constructs in NIH 3T3 cells was used to demonstrate that the increase in junB transcripts was specifically associated with v-src expression and could not be recapitulated with a c-src, v-H-ras, or v-raf expression vector. Deletion mutants were used to localize the v-src-responsive region in the junB promoter to a 121-nucleotide region encompassing the CCAAT and TATAA elements. This region is distinct from one in the 5' untranslated region of the junB gene which is required to maintain its high-level basal expression. Point mutagenesis of the junB TATAA box completely abolished v-src responsiveness, suggesting that proteins which bind to this element are modified by src transformation. Several v-src and c-src mutants were used to demonstrate that elevated tyrosine kinase activity of src proteins is required for the observed effects on junB expression. Finally, homology between the TATAA box regions of junB and the unrelated but src-responsive gene 9E3/CEF-4 suggests that modulation of gene activity through proteins which bind to this region may be a recurrent, although not exclusive, theme in src transforming action. Our results suggest that src proteins may modulate some nuclear effectors through pathways not involving cellular ras or raf gene products.
3T3 Cells↗
Modulation of cis-platinum resistance in Friend erythroleukemia cells by c-myc.
The molecular genetic basis for cancer cell resistance to the important family of platinum coordination complex drugs is poorly understood. The observation that malignant cells commonly become resistant to therapy, while normal cells rarely do, suggests that certain molecular processes involved in malignancy, e.g., oncogene activation, might also play a role in drug resistance. Since aberrant expression and amplification of various myc oncogenes have been implicated in the prognosis of human cancers, the poor therapeutic response of some of these tumors might be explained if high levels of myc gene products rendered cells more refractory to therapy. We tested this hypothesis by determining the effect of varying the level of c-myc expression on resistance to cis-platinum and ionizing radiation in Friend murine erythroleukemia cells expressing varying levels of c-myc gene product. We found that a) the degree of cis-platinum resistance correlated directly with the level of c-myc expression, b) glucocorticoid induction of murine mammary tumor virus promoter-driven c-myc sequences significantly increased cis-platinum resistance, c) restoring c-myc transcript levels to normal restored the original cis-platinum sensitivity at a rate which paralleled that of induced c-myc transcript depletion, and d) c-myc transcript level had no effect on ionizing radiation response. These findings suggest that c-myc levels may influence therapeutic success in some tumors and may regulate specific processes by which cells cope with DNA damage caused by DNA cross-linking agents such as the platinum analogues, but not ionizing radiation.
Cell Cycle↗
Depletion of c-myc with specific antisense sequences reverses the transformed phenotype in ras oncogene-transformed NIH 3T3 cells.
ras oncogene-transformed NIH 3T3 cells expressing glucocorticoid-inducible antisense c-myc cDNA transcripts at levels sufficient to deplete c-myc protein lost their transformed morphology and the ability to grow in soft agar; their ability to form tumors in nude mice was also impaired. These changes were dependent on the continuous expression of the antisense sequences. No major effects on plating efficiencies, growth rates in monolayer culture, or immortalization were observed in the revertant cells, indicating that the observed effects were not a toxic consequence of c-myc protein depletion. Transfection with the same vector expressing c-myc in the sense orientation or other control vectors had no effect on transformation. These results suggest that a certain minimum level of expression of c-myc is required for the maintenance of ras transformation in NIH 3T3 cells.
Animals↗
Antisense-mediated reduction in thrombospondin reverses the malignant phenotype of a human squamous carcinoma.
Thrombospondin (TSP) is a trimeric glycoprotein which is synthesized and incorporated into the extracellular matrix by a wide variety of cells. TSP is involved in a number of cellular processes which govern tumor cell behavior including mitogenesis, attachment, migration, and differentiation. To directly assess the role of TSP in tumor cell growth and spread, a human squamous carcinoma cell line, with high TSP production and an invasive phenotype, was transfected with a TSP cDNA antisense expression vector. Five unique transfected clones were obtained with reduced TSP production. Expression of the transfected antisense sequence in these clones was verified by a ribonuclease protection assay. These clones demonstrated reduced growth rates in vitro when compared with a vector transfected control. After subcutaneous inoculation into athymic mice, the antisense clones formed either no tumors or tumors that were slow growing and highly differentiated. This contrasted with the vector-transfected clone which produced poorly differentiated, rapidly growing, invasive tumors. Our results argue in favor of a direct role for TSP in determining the malignant phenotype of certain human tumors.
Animals↗
Amplified expression of three jun family members inhibits erythroleukemia differentiation.
Several different proto-oncogenes have been shown to influence cellular differentiation. One of the most widely studied model systems has been the Friend murine erythroleukemia cell (F-MELC) line, which can be induced to undergo erythroid differentiation by a variety of chemical agents. Constitutive overexpression of either the c-myc or c-myb proto-oncogenes has been previously shown to inhibit F-MELC differentiation, whereas c-myc antisense sequences accelerate the process. To investigate the potential involvement of other proto-oncogenes and immediate early response genes in F-MELC differentiation, we studied the expression of the three known members of the jun family as well as another gene, egr-1, which, like the jun family members, is expressed as an immediate early response gene in growth factor-stimulated quiescent cells. All four genes were expressed in F-MELC, although the levels of expression and modes of regulation differed. Transfection with amplifiable c-jun, junB, or junD expression plasmids inhibited differentiation, whereas transfection with an egr-1 expression plasmid was without effect. These results indicate that jun family members play a role in mediating F-MELC differentiation. The known inhibitory effect of phorbol ester tumor promoters on F-MELC differentiation may be the result of their known stimulation of jun expression.
Cell Line↗
Tumor necrosis factor-alpha induction of novel gene products in human endothelial cells including a macrophage-specific chemotaxin.
Cytokines such as tumor necrosis factor alpha (TNF) profoundly affect endothelial cell function, promoting for example interaction with leukocytes and inducing a procoagulant phenotype. Changes of this nature are likely to be central to the proinflammatory effects of TNF. In order to elucidate molecular mechanisms by which TNF alters endothelial cell function we utilized differential plaque hybridization to identify TNF-responsive genes. Forty TNF-inducible cDNAs were identified which on cross-hybridization were found to arise from six unique genes. DNA sequencing of these cDNAs revealed two encoded known cytokine-induced genes, endothelial leukocyte adhesion molecule 1 and neutrophil chemotactic factor. One of the cDNAs encodes a recently described monocyte-specific chemotactic factor not previously associated with endothelium. The production of a monocyte chemotaxin by cytokine-activated endothelium has important implications for understanding the role of the vessel wall in disease states such as atherosclerosis and may also in part explain the indirect angiogenic activity of TNF. The three other cDNAs are completely novel as judged by data bank searches of partial DNA sequences and remain unidentified. On exposure of endothelial cells to TNF there is a rapid and substantial increase in levels of mRNA encoding the six genes, which are further superinduced by cycloheximide. Thus these represent primary response genes as their induction does not depend on protein synthesis. Interleukin-1 beta and lipopolysaccharide are also potent inducers. Nuclear run-on studies revealed that in most cases induction by TNF is mediated largely at the transcriptional level.
Biological Factors↗
Embryonal rhabdomyosarcoma of the ampulla of Vater with long-term survival following pancreaticoduodenectomy.
Rhabdomyosarcoma of the biliary tree is a rare cause of biliary tract obstruction in childhood. A 3-year-old child is reported here after presenting with obstructive jaundice secondary to an embryonal rhabdomyosarcoma of the ampulla of Vater. He underwent pancreaticoduodenectomy followed by adjuvant chemotherapy and irradiation. He is now well and free of disease 5 years following treatment. This child appears to be the first long-term survivor who has required pancreaticoduodenal resection for this lesion.
Ampulla of Vater↗
The leucine zipper of c-Myc is required for full inhibition of erythroleukemia differentiation.
The leucine zipper motif has been observed in a number of proteins thought to function as eucaryotic transcription factors. Mutation of the leucine zipper interferes with protein dimerization and DNA binding. We examined the effect of point mutations in the leucine zipper of c-Myc on its ability to dimerize in vitro and to inhibit Friend murine erythroleukemia (F-MEL) differentiation. Glutaraldehyde cross-linking studies failed to provide evidence for homodimerization of in vitro-synthesized c-Myc protein, although it was readily demonstrated for c-Jun. Nevertheless, whereas transfected wild-type c-myc sequences strongly inhibited F-MEL differentiation, those with single or multiple mutations in the leucine zipper were only partially effective in this regard. Since the leucine zipper domain of c-Myc is essential for its cooperative effect in ras oncogene-mediated transformation, this study emphasizes the close relationship that exists between transformation and hematopoietic commitment and differentiation. c-Myc may produce its effects on F-MEL differentiation through leucine zipper-mediated heterodimeric associations rather than homodimeric ones.
Amino Acid Sequence↗