Consequences of antisense human chorionic gonadotrophin-α subunit cDNA expression in human choriocarcinoma JAR cells

Abstract

The biosynthesis of human chorionic gonadotrophin (hCG) is a hallmark endocrine function of human choriocarcinoma cells. The present study investigated the consequences of greatly diminishing this synthesis in JAR cells by stably transfecting them with pRSV-antisense hCG-alpha cDNA expression vector. The vector directs the synthesis of antisense hCG-alpha subunit mRNA which would then bind to sense hCG-alpha subunit mRNA, thus blocking its translation and consequently dimer hCG protein synthesis. The transfection with pRSV-antisense hCG-alpha cDNA resulted in a dramatic decrease in hCG secretion as compared with untransfected parental cells or those transfected with an empty vector used for the selection of clones. The decreased secretion was due to a decreased synthesis which in turn was due to a fall in steady-state hCG-alpha and -beta subunit mRNA levels. The decrease of hCG-beta subunit transcripts was unexpected and it was not due to contamination of antisense hCG-alpha cDNA construct with hCG-beta sequence. The transcription of hCG-alpha and -beta subunit genes was not altered in transfected cells suggesting that increased degradation was responsible for decreased steady-state hCG subunit mRNA levels. Despite the decreased hCG levels, the transfected cells maintained normal hCG receptor levels, responded to epidermal growth factor stimulation of hCG synthesis and secretion and grew at the same rate as the control parental cells and those transfected with an empty vector.

Transfection of Antisense Chorionic Gonadotropin β Gene into Choriocarcinoma Cells Suppresses the Cell Proliferation and Induces Apoptosis

“…There is a possibility that high hCG ␤-subunit concentrations could result in the formation of homodimers that could potentially mimic heterodimers due to conformation similarity. A reduction in hCG synthesis was found to have no effect on cell proliferation in human choriocarcinoma JAr cells (24). Furthermore, Lei et al (36) demonstrated that the tumorigenic effects of hCG did not include an increase in cell proliferation, but, rather, decreased apoptosis and increased the ability of cell invasion of JAr cells in nude mice.…”

“…The introduction of recombinant plasmid expression vectors that direct synthesis of antisense mRNA has been performed to inhibit the translation of sense mRNA in different cells in vitro (22)(23)(24)(25)(26)(27). Using this procedure, either reduction of endogenous protein synthesis (22)(23)(24)(25) or cellular growth inhibition was achieved in certain cells (26,27).…”

“…Using this procedure, either reduction of endogenous protein synthesis (22)(23)(24)(25) or cellular growth inhibition was achieved in certain cells (26,27). In this context, the present study was conducted to ascertain whether the transfection of antisense CG␤ gene into choriocarcinoma cells affects proliferation and apoptosis in those cells.…”

How and Why Do Gestational Trophoblastic Neoplasms Overproduce Human Chorionic Gonadotropin?

“…If this were to be the case, inhibition of hCG biosynthesis will have consequences to choriocarcinoma cells. To test this possibility, hCG synthesis was inhibited by stable transfection with antisense of gonadotropin-α subunit cDNA [16]. The antisense α-subunit transcripts made will dimerize with protein coding sense α-subunit transcripts and the dimers formed will then be rapidly degraded by nucleases.…”

Gene Therapy of Glioblastoma: Anti – Gene Anti IGF-I Strategy

“…ASODN may provide a novel approach to therapy of gastric cancer. The antisense technology was used to study several protein actions: the alpha subunit of human chorionic gonadotrophin in choriocarcinoma cells (Cao et al, 1995); the regulating protein E2F-1, in S cellular cycle phase, and its action on genes linked to proliferation (Sala et al, 1994); nerve growth factor (NGF) in skin of transgenic mice, and its relationship with response to mechanical stimuli (Davis et al, 1993). Lately, the antisense strategy is "classically" used to analyze gene expression and intron splicing.…”