Promotion of growth and apoptosis in c-myc nullizygous fibroblasts by other members of the myc oncoprotein family

Abstract

c-myc nullizygous fibroblasts (KO cells) were used to compare the abilities of c-myc, N-myc and L-myc oncoproteins to accelerate growth, promote apoptosis, revert morphology, and regulate the expression of previously described c-myc target genes. All three myc oncoproteins were expressed following retroviral transduction of KO cells. The proteins all enhanced the growth rate of KO cells and significantly shortened the cell cycle transition time. They also accelerated apoptosis following serum deprivation, reverted the abnormal KO cell morphology, and modulated the expression of previously described c-myc target genes. In most cases, Lmyc was equivalent to c-myc and N-myc in restoring all of the c-myc-dependent activities. These findings contrast with the previously reported weak transforming and transactivating properties of L-myc. Myc oncoproteins may thus impart both highly similar as well as dissimilar signals to the cells in which they are expressed. Cell Death and Differentiation (2000) 7, 697 ± 705.

Functional analysis of the N-terminal domain of the Myc oncoprotein

“…To investigate the nature of the role of this amino acid, we mutated it to a conserved, nonpolar or charged amino acid (W135F, W135A and W135E, respectively). We also included the Myc family members N-Myc and LMyc in our study as they are highly homologous to cMyc (39 and 33% amino-acid identity, respectively) and appear to have overlapping, although not completely, redundant functions (DePinho et al, 1987;Small et al, 1987;Birrer et al, 1988;Barrett et al, 1992;Nesbit et al, 1998;Landay et al, 2000). For the majority of our studies, we employed the Myc-null HO15.19 cell line, which lacks endogenous expression of c-, N-and L-Myc proteins (Mateyak et al, 1997) Figure 2).…”

Onzin, a c-Myc-repressed target, promotes survival and transformation by modulating the Akt–Mdm2–p53 pathway

“…For some experiments, a CMV-based expression vector containing a similarly amplified onzin cDNA fused at its 5 0 -end to a FLAG epitope in the pCMV2-FLAG vector (Eastman Kodak, Inc., Rochester, NY, USA) was used. For expression of onzin in primary fibroblasts, the Myc epitope-tagged onzin cDNA was amplified from the pSVL-(MT)puro vector and cloned into the pMN-IRES-GFP retroviral vector (Landay et al, 2000). For stable expression of Akt in Rat1a cells, the complete coding region of Akt was fused with the c-Myc epitope tag in the pSVL-(MT)puro vector.…”

“…The indicated Rat1a-onzin or Rat1a-puro clones were plated at 10 5 cells/well, grown to 20-30% confluence, and then either deprived of serum or exposed to fresh medium containing VP-16 or adriamycin. Viable cell counts were then performed daily using trypan blue exclusion as described previously (Nesbit et al, 1998;Landay et al, 2000). Ongoing apoptosis was documented by showing the presence of cells with a subdiploid DNA content and by the TUNEL assay as described previously (Mu et al, 2002; data not shown).…”

“…None of these expressed detectable onzin transcripts as determined by Northern blotting (Figure 1b; data not shown). Retroviral transduction was used to obtain pooled populations of both human strains (Landay et al, 2000), whereas several clonal isolates of Rat1a cells were obtained following standard transfection with a nonretroviral onzin expression vector (Figure 3a). In all three cases, control transfections were performed in parallel with vectors lacking onzin sequences.…”

“…Fibroblasts were infected overnight with retroviral supernatants in the presence of 8 mg/ml polybrene. At 3-4 days after infection, GFP-positive cells were purified by cell sorting, expanded as described previously (Landay et al, 2000), and utilized for all subsequent studies.…”