Ornithine decarboxylase over-expression stimulates mitogen-activated protein kinase and anchorage-independent growth of human breast epithelial cells.
In these experiments we tested the hypothesis that constitutive activation of polyamine(PA) biosynthesis may contribute to mammary carcinogenesis. Spontaneously immortalized normal human MCF-10A breast epithelial cells were infected with the retroviral vector pLOSN containing a cDNA which codes for a truncated and more stable ornithine decarboxylase (ODC), the rate-limiting enzyme in PA synthesis. Upon chronic selective pressure with alpha-difluoromethyl-ornithine (DFMO) (an irreversible inhibitor of ODC), infected MCF-10A cells exhibited an approximately 250-fold increase in ODC activity, which persisted despite discontinuation of DFMO. ODC-over-expressing MCF-10A cells showed a modest decrease in S-adenosylmethionine decarboxylase and an increase in spermidine/spermineN1-acetyltransferase. Analysis of cellular PA profile revealed a selective accumulation of putrescine without alterations in spermidine and spermine contents. Lesser degrees of increased ODC activity were obtained reproducibly by re-exposing the cells to incremental small doses of DFMO. We observed a bell-shaped dose-related positive effect of ODC activity on clonogenicity in soft agar of MCF-10A cells. Since anchorage-dependent growth was actually reduced, such positive influence on this feature of transformation was not a non-specific consequence of a growth advantage provided by ODC over-expression. In addition, we observed a close parallelism between the dose-dependent effects of ODC expression on clonogenicity and activity of the ERK-2 kinase, a central element of the MAPK cascade. Our data demonstrate an interaction between PA and the MAPK signalling pathway and suggest that the latter may be involved in ODC-induced transformation of mammary epithelial cells.
Manni
A
A
Department of Medicine, Milton S. Hershey Medical School, Pennsylvania State University, Hershey, USA.
Wechter
R
R
Gilmour
S
S
Verderame
M F
MF
Mauger
D
D
Demers
L M
LM
D000123:Acetyltransferases / Q000378:metabolism; D012437:Adenosylmethionine Decarboxylase / Q000378:metabolism; D001940:Breast / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002461:Cell Line, Transformed; D002471:Cell Transformation, Neoplastic / Q000235:genetics*; D003114:Colony-Forming Units Assay; D018076:DNA, Complementary / Q000235:genetics; D000518:Eflornithine / Q000494:pharmacology; D004790:Enzyme Induction; D004847:Epithelial Cells; D004848:Epithelium / Q000187:drug effects / Q000201:enzymology; D005260:Female; D011905:Genes, ras; D006801:Humans; D019950:Mitogen-Activated Protein Kinase 1; D009955:Ornithine Decarboxylase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D065108:Ornithine Decarboxylase Inhibitors; D011073:Polyamines / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D014162:Transfection; D000099228:Diamine N-Acetyltransferase
D000123:Acetyltransferases; D012437:Adenosylmethionine Decarboxylase; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D018076:DNA, Complementary; D000518:Eflornithine; D019950:Mitogen-Activated Protein Kinase 1; D009955:Ornithine Decarboxylase; D065108:Ornithine Decarboxylase Inhibitors; D011073:Polyamines; D011993:Recombinant Fusion Proteins; D000099228:Diamine N-Acetyltransferase