Abstract
A spontaneous transformant derived from a mouse lung epithelial cell line exhibited decreased cAMP-dependent protein kinase (PKA) activity. DEAE column chromatography demonstrated that this was caused by specific loss of the type I PKA isozyme (PKA I). Western immunoblot analysis indicated that indeed several mouse lung tumor-derived cell lines and spontaneous transformants of immortalized, nontumorigenic lung cell lines contained less PKA I regulatory subunit (RI) protein than normal cell lines. PKA II regulatory subunit protein differed only slightly among cell lines and showed no conspicuous trend between normal and neoplastic cells. The decrease in RI was apparently concomitant with decreased catalytic (C) subunit levels in neoplastic cells since no free catalytic subunit activity was detected by DEAE chromatography. Northern blot analysis using RI alpha and C alpha cDNA probes showed that the levels of RI alpha and C alpha mRNAs paralleled their intracellular protein concentrations; neoplastic cell lines contained significantly less RI alpha and C alpha mRNAs than the normal cell line. The decreased expression of both RI and C subunits therefore results in a net decrease of PKA I in neoplastic lung cells, an isozymic difference which may account for the differential effects of cAMP analogs on cell growth and differentiation in normal and neoplastic cells.
Highlights
Decreased Expression of the Type I Isozyme of CAMP-dependent Protein Kinase in Tumor Cell Lines of Lung Epithelial Origin*
Western immunoblot analysis indicated that several mouse lung tumor-derived cell lines and spontaneous transformants of immortalized, nontumorigenie lung cell lines contained less PKA I regulatory subunit (RI) protein than normal cell lines
Was apparently concomitant with decreased catalytic (C) subunit levels in neoplastic cells since no free catalytic subunit activity was detected by DEAE chromatography
Summary
Cell Culture-The immortalized nontumorigenic cell lines (hereafter referred to as “normal” cell lines) NallA C4ElO (E10) and. Medical Center, Brooklyn, NY), which was derived from an alveolar tumor induced in Swiss-Webster mice following transplacental carcinogenesis with ethylnitrosourea; and PCC4 Postmitochondrial supernatant (cytosolic) fractions were prepared by a IO-min centrifugation of 20,000 x g, and the recovered pellet was resuspended in the original volume of buffer. Chromatography-Cytosolic fractions containing 5-8 mg of protein were preincubated in 0.15 mM ATP and 3.5 mM MgC12 at 30 “C for 30 min to facilitate association of the PKA I holoenzyme and passed over a Sephadex G-25 column (30 X 0.9 cm). Preparation of Total RNA and Northern Analysis-Cells were washed three times with fresh pre-chilled media, scraped into fresh media, pooled, and centrifuged at 135 x g for 5 min.
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