Isolation of antibodies specific for avian viral and cellular myc proteins

The myc gene has been implicated in the genesis of various neoplasms in birds, mice, and humans and was originally identified as the cellular homologue of the transforming gene (v-myc) of the avian myelocytomatosis virus MC29. For specific antisera to be obtained for the myc gene product, a bacterial expression vector was constructed in which the coding sequences for approximately 20 kd of MC29 p110gag-myc (amino acid residues 502 to 678) were placed between the coding sequences for the amino terminal 13 kd of Rous sarcoma virus pp60src and the coding sequences for 112 kd of beta-galactosidase. Expression of this tripartite gene was driven by a hybrid trp-lac promoter under lac repressor control. Induction of expression resulted in the production of a 145-kd hybrid protein containing src, myc, and beta-galactosidase sequences. The hybrid protein was purified and injected into rabbits to produce antisera. The resultant antisera immunoprecipitated p110gag-myc and p58myc -p60myc from MC29- and MH2-infected nonproducer quail fibroblasts, respectively. In addition, the antisera also immunoprecipitated a 58-kd protein from the bursal lymphoma cell line BK25, which was identified as chicken c (cellular)-myc gene product.     

Association of v-myc protein with chromatin

The subnuclear distribution of proteins encoded by v-myb and v-myc was analysed in a cell-line of AMV-transformed chicken myeloblasts superinfected by the myc-containing retrovirus MC29. p45v-myb and p110gag-myc, co-expressed in these cells, were released in similar fashion when nuclei were treated with salt or DNAase. Analysis of nucleoprotein complexes extracted from nuclease-treated nuclei shows that p45v-myb and p110gag-myc are associated with a chromatin fraction of enhanced nuclease sensitivity. v-myb and v-myc proteins thus share the same subnuclear location and apparently interact directly with the cellular DNA.     

Myc protein structure: localization of DNA-binding and protein dimerization domains

Wildtype and mutant v-Myc proteins were overexpressed in Escherichia coli using the T7 RNA polymerase system, and the in vitro DNA-binding activities of partially or highly purified proteins were analysed by native DNA-cellulose chromatography. For the construction of the expression plasmids, cloned proviral DNA from wildtype MC29 or from its spontaneous deletion mutant Q10C was used, the latter lacking internal v-myc sequences. Both the wildtype (p59) and the mutant (p42) recombinant protein contain at their amino termini 12 amino acids encoded by the vector, followed by 11 gag amino acids and 9 amino acids encoded by v-myc sequences derived from noncoding c-myc sequences. In addition, p59 contains 416 amino acids encoded by v-myc sequences derived from the complete chicken c-myc coding region, whereas p42 lacks 120 amino acids from the central region of the Myc protein including the highly acidic domain. Two additional proteins were engineered which contain the first 309 (p53) or the last 107 (p16) amino acids, respectively, of the Myc protein sequence in addition to vector-encoded amino acids. The p16 protein represents the carboxyl terminus of the Myc protein sequence containing both a muscle determination gene (MyoD1) homology region, including a basic motif and an amphipathic helix-loop-helix motif, and a leucine heptad repeat. All proteins, except p53 which lacks the carboxyl-terminal Myc protein sequences, bound to native DNA-cellulose and were eluted with 200-500 mM NaCl. Based on the DNA-binding activities of recombinant or spontaneous mutant v-Myc proteins extracted from bacterial or from transformed avian cells, we conclude that the DNA-binding domain of avian Myc proteins is confined within the last 86 carboxyl-terminal amino acids. The same region is also shown to be necessary and sufficient for Myc protein dimerization. This 86-amino acid region essentially encompasses a putative basic DNA contact surface and a tandem array of two presumed protein dimerization motifs, helix-loop-helix and leucine repeat.     

Transformation of murine myelomonocytic cells by myc: point mutations in v-myc contribute synergistically to transforming potential

The v-myc oncogenes of chicken retroviruses (including MC29) bear point mutations relative to chicken c-myc. These mutations result in several amino acid differences in the encoded proteins. We have used recombinant murine retroviruses containing various myc alleles to analyse the myelomonocytic transforming potential of the myc oncogene. The myc alleles used were MC29 v-myc, chicken c-myc, chimeric genes combining 5' sections of v- or c-myc with 3' sections of c- or v-myc, and mouse c-myc. The same retroviral vector (based on the genome of Moloney leukemia virus) was used for each allele and the genes were translated from genomic message. By infecting the primary mouse tissues, bone marrow, peritoneal-derived macrophages and mixed embryonic tissue with the recombinant viruses, variation was found in the transforming efficacy of these alleles: v-myc was most effective, followed by the two chimeric genes, whereas c-myc (chicken or mouse) was least effective in eliciting myelomonocytic transformation. Viral gag sequences were not necessary for this transformation. In each case, the transformed monocytes were growth factor-dependent and non-immortal. However, v-myc transformed monocytes (though not monocytes transformed by other myc alleles) were able to progress to an immortal, growth factor-independent phenotype. Our results indicate that v-myc is far more effective than c-myc in eliciting myelomonocytic transformation; that this is due to combinatorial effects of 5' and 3' mutations in the v-myc gene; and that secondary events in addition to these mutations are required for transformation of myelomonocytic cells to an immortal, tumorigenic phenotype.     

The transforming activity of the chicken c-myc gene can be potentiated by mutations

It was previously demonstrated that four different avian v-myc oncogenes harbor several point mutations. At least one of these leads to an amino acid substitution located in the proximity of position 61 in the second exon, whereas additional substitutions are found in exon 3. We have investigated whether these mutations affect the transforming activity of myc. By constructing avian retroviral genomes expressing hybrid gag-myc oncogenes, in which all or parts of the v-myc domains were replaced by corresponding parts of c-myc, we show here that a substitution of threonine 61 of c-myc for a methionine (as in v-mycmc29) significantly enhances the fibroblast transforming capacity of the recombinant oncogene. However, such a hybrid v/c-myc gene is still several fold less active than the v-mycmc29 oncogene. We have also expressed c-myc from subgenomic retroviral mRNAs: in these constructions the AUG of gag in the RNA leader sequence is in the same reading frame as that of c-myc, apparently leading to the production of a myc protein with 11 N-terminal amino acids encoded by gag and non-coding c-myc sequences. These myc proteins also transform chicken embryo fibroblasts, albeit with a lower efficiency than v-myc, again suggesting that mutations can increase the transforming capacity of myc.     

Amplification of myc-specific sequences in human colonic cancer

DNA samples obtained from tumors and adjacent mucosa of the large bowel of a patient with large bowel multiple neoplasia were examined after Southern. The procedure established amplification of v-myc oncogene-related DNA sequences in 1 out of 5 tumors tested. Restriction fragments of amplified myc-specific sequences and matching c-myc and N-myc loci of the human genome differed in size.     

Oncogene-mediated multistep transformation of C3H10T1/2 cells

We have examined the response of the mouse embryonic cell line C3H10T1/2 to transfection with the activated human c-H-ras oncogene and the gag-myc oncogene from avian myelocytomatosis virus 29. C3H10T1/2 cells are not morphologically transformed following transfection with the gag-myc oncogene. A low level of focus formation is observed following transfection of the c-H-ras oncogene. When C3H10T1/2 cells are cotransfected with the ras and myc oncogenes, focus formation is increased by an average of 13 fold. In addition, C3H10T1/2 ras/myc foci have a distinct, transformed morphology which correlates with an increased potential for anchorage-independent growth. Although morphological transformation in this system is largely a function of ras oncogene expression, our studies demonstrate that it is potentiated by the presence of a functional gag-myc protein. Oncogene-mediated multistep transformation, which was first described in primary embryo cultures, is not a general property of established cell lines. The C3H10T1/2 cell line is an exception and provides a model system in which partially transformed phenotypes, in a progression toward malignant transformation, can be isolated and studied.     

Inducible transformation of fibroblasts using a metallothionein-v-myc gene construct

An inducible oncogene construct has been engineered by coupling the MC29 v-myc oncogene to the sheep metallothionein promoter. Transfection of this plasmid, which also contains the neomycin resistance gene, into Rat-1 cells, has resulted in the isolation of independent clones resistant to G418. Certain of these clones were found to exhibit inducible transformation in response to ZnSO4. Transformation was graded with increasing ZnSO4 levels and was reversible when ZnSO4 was removed from the media. By analyzing v-myc mRNA levels, the inducible alterations in cellular morphology and growth were found to be associated with increased v-myc expression. The metallothionein promoter exhibited negligible constitutive expression of v-myc and none of the clones isolated exhibited spontaneous transformation. Our results show that the use of a metallothionein promoter v-myc construct facilitates the study of inducible fibroblast transformation.     

Cooperative effect of v-myc and v-erbA in the chick embryo.

We show that a construct designated as MAHEVA, which encodes oncogenes v-myc from MH2 virus and v-erbA from AEV under the control of the LTR of MH2, induces rapidly growing heart rhabdomyosarcomas, when it is injected in E3 but not E5 chick embryos. A similar pathology has previously been observed with MC29, within the same limited time frame. The tumors, which expressed P61-63myc, P75gag-erbA and Pr76gag proteins were detectable from E14 onwards. Compared with MC29, MAHEVA induced a secondary anomaly, not detectable prior to E17. This is the appearance of cartilage nodules within the heart rhabdomyosarcomas. The constant location of these nodules inside the rhabdomyosarcomas and their delayed appearance suggests that the chondrocytes originate from myoblasts prevented from differentiating by the expression of the v-myc product. This interpretation is supported by the appearance of chondrocytes in E3 heart muscle cells infected in vitro with MAHEVA.     

Tumor progression following transformation of murine monocytes by v-myc: acquisition of immortalization and tumorigenicity.

Monocyte transformation by the v-myc oncogene has been used to study myelomonocytic tumor progression in vitro. Murine monocytes transformed by a recombinant retrovirus containing MC29 v-myc were found to exhibit a proliferative burst to day 28-40 post-infection. There-after growth slowed and cell number remained relatively static to day 80-90 post-infection. During both the proliferative and quiescent periods, the cells were dependent on the myelomonocytic growth factor CSF-1 for growth and viability. Analysis of this transformation revealed that the initial transformants were polyclonal, non-immortal, and non-tumorigenic in syngeneic mice. At day 80-90 post infection, a fresh round of cellular proliferation occurred and, in contrast to the initial burst, growth was sustained allowing the establishment of cell lines. These lines were found to be monoclonal, immortal, growth factor independent and, in certain cases, tumorigenic in syngeneic mice. Associated with the establishment of growth factor independent cell lines was the constitutive synthesis of the myelomonocytic growth factor, CSF-1. Proto-oncogene screening of the initial transformants and the cell lines also revealed the expression of c-raf and the CSF-1 receptor, c-fms. Our results indicate that, following transformation by v-myc, monocytes can progress in vitro to become growth factor independent and immortal and that both monocyte transformation and immortalization can be dissociated from tumorigenicity.     

Constitutive expression of exogenous myc in myelomonocytic cells: acquisition of a more transformed phenotype and inhibition of differentiation induction.

The effects of deregulated expression of the human c-myc and MC29 v-myc oncogenes have been examined in a murine myelomonocytic cell line J774 (c-myc) and in a variety of myelomonocytic cell lines of different degrees of maturity generated from primary hematopoietic tissue (v-myc). Introduction of a Moloney murine leukemia virus long terminal repeat (LTR) c-myc construct into J774 cells resulted in constitutive expression of the exogenous myc gene and a concomitant increase in the degree of transformation and tumorigenicity of the cells. In addition, constitutive expression of exogenous myc inhibited induced differentiation of these cells by a variety of treatments including addition to the medium of lipopolysaccharide (LPS) or the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA) as well as complete withdrawal of serum from the medium. The degree of increased transformation, tumorigenicity and inhibition of terminal differentiation was dependent upon the level of exogenous myc expression. For the v-myc-generated myelomonocytic cell lines, introduction of v-myc resulted in a high degree of transformation and, irrespective of the differentiation status of the cells, a block of induced differentiation. These results indicate that the level of constitutive myc expression can affect the transformed phenotype, tumorigenicity and differentiation inducibility of myelomonocytic cells.     

Mutant p53 can provoke apoptosis in p53-deficient Hep3B cells with delayed kinetics relative to wild-type p53

Wild-type (wt) p53 frequently induces apoptosis when expressed in tumor cells whereas mutant p53 acts as an oncoprotein and consequently, stimulates cell proliferation. We report here exceptions to that rule. p53 conformational mutant 175H and DNA contact mutant 273H provoke apoptosis in human p53-deficient Hep3B hepatoma cells with delayed kinetics relative to wt p53. Similarly, c-Myc strongly stimulates apoptosis in these cells. In contrast, viral oncoproteins E1A and E7, and the cellular oncoprotein MDM-2, fail to elicit cytocidal responses. Efficient apoptotic cell death by mutant p53 requires oligomerization as 175H and 273H with deletions between amino acid residues 326 and 347 of the oligomerization domain are nontoxic. Apoptosis by mutant or wt p53 was significantly inhibited by the serine protease inhibitor AEBSF but not by the inactive analog AEBSA. Together, these results suggest that a wt p53-independent control mechanism is operational in Hep3B cells that eliminates cells upon sensing illegitimate proliferation signals originating from certain oncoproteins, including mutant p53 and Myc. We suggest that some tumor cell types lack p53 altogether because they tolerate neither wild-type nor mutant forms of the protein.     

The leucine zipper domain of avian cMyc is required for transformation and autoregulation

Small deletions of 7 to 48 amino acids have been generated in the leucine zipper domain of the avian cMyc protein and the mutant cMyc proteins expressed using an avian retroviral vector. Retrovirally encoded cMyc protein transforms primary chick embryo fibroblasts and leads to abnormal regulation of the endogenous c-myc gene. Deletion of the most C-terminal leucine of the zipper motif confers a partial phenotype affecting some but not all parameters of transformation. Complete loss of transforming activity results from deletion of further leucine residues, including one which is not part of the heptad repeat. In cMyc transformed cells endogenous c-myc mRNA is expressed at a low level and is abnormally refractory to induction by serum stimulation. In contrast, a non-transforming cMyc protein which lacks the zipper does not affect normal c-myc expression. These results demonstrate that the leucine zipper domain of avian cMyc is required for both transformation and autoregulation, and suggests that essential leucine residues within the motif may be spaced differently from those in the zippers of Fos and Jun.     

Nuclear localization is essential for the activity of p53 protein.

p53 appears to be a growth regulator, the perturbation of which induces changes in normal cell proliferation. Wild-type p53 protein is thought to function as a growth arrest gene, whereas mutant p53, which accumulates in transformed cells, has been shown to enhance malignant transformation. Both wild-type and mutant p53 migrate into the cell nucleus by means of identical nuclear localization signals (NLS) inherent in their primary sequences. Results presented here show that the suppressive activity of wild-type p53 measured as the reduction of transformation of primary rat fibroblasts induced by co-transfection with ras and either E1A or mutant p53, as well as the transformation enhancement of mutant p53 estimated by cooperation with ras in transformation of primary rat fibroblasts, is dependent upon nuclear localization signals in p53 protein. While transfection of unmodified wild-type p53 significantly reduces the number of rat embryonic fibroblast-transformed foci induced by E1A and ras or mutant p53 and ras, the wild-type p53 protein without NLS has completely lost this suppressive activity. Partially defective NLS wild-type p53, with a reduced nuclear accumulation ability, still exhibits some suppressive activity. In addition, we found that plasmids coding for intact mutant p53 protein efficiently cooperate with the ras oncogene, whereas the corresponding plasmids without NLS are totally inert. On this basis we conclude that nuclear localization of both wild-type and mutant p53 is a fundamental feature for manifesting the activities of these proteins. Both the suppressor activity mediated by the wild-type p53 and enhancement of transformation mediated by the mutant p53 require nuclear localization of the proteins to function.     

Phosphorylation of histone H3 at serine 10 is indispensable for neoplastic cell transformation

Very little is known about the role of histone H3 phosphorylation in malignant transformation and cancer development. Here, we examine the function of H3 phosphorylation in cell transformation in vivo. Introduction of small interfering RNA-H3 into JB6 cells resulted in decreased epidermal growth factor (EGF)-induced cell transformation. In contrast, wild-type histone H3 (H3 WT)-overexpressing cells markedly stimulated EGF-induced cell transformation, whereas the H3 mutant S10A cells suppressed transformation. When H3 WT was overexpressed, EGF induction of c-fos and c-jun promoter activity was significantly increased compared with control cells but not in the H3 mutant S10A or S28A cells. In addition, activator protein-1 activity in H3 WT-overexpressing cells was markedly up-regulated by EGF in contrast to the H3 mutant S10A or S28A cells. These results indicate that the phosphorylation of histone H3 at Ser10 is an essential regulatory mechanism for EGF-induced neoplastic cell transformation.     

Expression of an amino-terminal BRCA1 deletion mutant causes a dominant growth inhibition in MCF10A cells

Expression of deletion mutants of the breast and ovarian cancer-specific tumor suppressor protein, BRCA1, in the mammary epithelial cell line MCF10A revealed a powerful growth suppressive effect by a mutant that has the amino-terminal 302 amino acids deleted (DeltaN-BRCA1). The growth suppression is associated with an increase in apoptosis and amplification in centrosome number. The growth inhibitory effect of DeltaN-BRCA1 was not observed in cervical epithelial HeLa cells, suggesting that the phenotypes of BRCA1 mutant proteins differ depending on the cell line being tested. An internal domain, including BRCA1 residues 303-1292, caused the suppression of MCF10A cell growth, and the amino terminus of BRCA1 autoinhibited the growth suppression. Single point mutations that disrupted the amino-terminal RING domain of BRCA1 caused significant suppression of growth in MCF10A cells. These results suggest that the proper function of the RING domain, likely to be ubiquitin ligase function, is important in regulating the growth of the mammary epithelial cell line and in autoregulating the powerful internal growth-inhibiting domain of the BRCA1 tumor suppressor.