Induction of cell transformation by mutated 16K vacuolar H+-atpase (ductin) is accompanied by down-regulation of gap junctional intercellular communication and translocation of connexin 43 in NIH3T3 cells.
The 16 K subunit of the vacuolar H+-ATPase (ductin) has been suggested to also play a role in gap junction channels. Since mutated 16 K subunits have transforming ability when transfected into NIH3T3 cells and since aberrant gap junctional intercellular communication (GJIC) is a hallmark of cancer cells, we hypothesized that mutated 16 K subunits might transform these cells via alteration of GJIC. When GJIC was measured by the dye-transfer assay, NIH3T3 cells transfected with the mutant 16 K protein genes (deletion of the fourth transmembrane domain or a point mutation at codon 143 from glutamic acid to arginine) showed significantly lower levels of GJIC than those transfected with the vector alone or with the wild-type 16 K subunit gene. GJIC levels of NIH3T3 cells transformed by v-ras and v-src were not significantly decreased, suggesting that low GJIC levels are not necessarily the result of cell transformation per se. NIH3T3 cells express C x 43 as a major connexin gene. Although cells transfected with mutated 16 K subunits showed a level of C x 43 protein expression similar to non-transfectants, their C x 43 protein was localized aberrantly, i.e. intracytoplasmically. These results indicate that mutant 16 K subunits with transforming ability translocate C x 43 proteins, thus inhibiting GJIC of NIH3T3 cells.
Saito
T
T
Unit of Multistage Carcinogenesis, International Agency for Research on Cancer, Lyon, France.
Schlegel
R
R
Andresson
T
T
Yuge
L
L
Yamamoto
M
M
Yamasaki
H
H
D016475:3T3 Cells; D000818:Animals; D001692:Biological Transport; D002471:Cell Transformation, Neoplastic; D018031:Connexin 43 / Q000235:genetics / Q000378:metabolism*; D015536:Down-Regulation; D017629:Gap Junctions / Q000378:metabolism*; D015870:Gene Expression; D007424:Intracellular Fluid; D051379:Mice; D016296:Mutagenesis; D009857:Oncogenes; D011510:Proteolipids / Q000235:genetics / Q000378:metabolism*; D006180:Proton-Translocating ATPases / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger; D015398:Signal Transduction; D014162:Transfection; D025262:Vacuolar Proton-Translocating ATPases
D018031:Connexin 43; D011510:Proteolipids; D012333:RNA, Messenger; C082471:ductin; D025262:Vacuolar Proton-Translocating ATPases; D006180:Proton-Translocating ATPases