In vitro synthesis and secretion of albumin by Morris hepatomas 5123C and 7800.

INTRODUCTION Morris hepatomas Si 23C and 7800 were incubated in vitro with radioactive L-leucine in Krebs-Ringer-bicar bonate-saline, and the incorporation of radioactivity into albumin precursors, serum albumin, and tnichioroacetic acid-precipitable proteins was measured and compared to that which occurs in normal rat liver slices. At the end of a 10-mm pulse incubation with radioactive L-Ieucine, Morris hepatoma 7800 incorporated 0.25% of the total protein radioactivity into an albumin fraction while Morris Hepa toma 5i23C incorporated 0.5% and rat liver slices showed a much larger proportion of incorporation (4.8%) into albu mm. The pulse-labeled tissues were further incubated with nonradioactive L-ieucine for different periods of time, and they all released radioactive proteins into the incubation medium. Only 5% of the total protein radioactivity released into the medium by Morris hepatoma 7800 and 7.8% of that released by Morris hepatoma 5i23C were incorporated into albumin while, in contrast, rat liver slices released 46% of its protein radioactivity as albumin. Both hepatomas were capable of releasing 80% of the pulse-labeled albumin into the medium at the end of a 75-mm chase period. Analyses of the nascent albumin within the tissues of Morris hepato mas 7800 and 5123C and in rat liver slices showed that, at the end of a 10-mm pulse incubation, the intracellular albumin fraction could be identified as a precursor of serum albumin (proalbumin) and that, as the chase incubation proceeded, proalbumin was converted intracellulanly to another form of albumin which was chromatographically distinct from serum albumin. The radioactive albumin which was released into the medium, however, closely resembled serum albumin. Like normal liver, Morris hepa tomas 5123C and 7800 are able to segregate nascent proalbumin in the microsomal cell fraction, and only a small amount (8%) was found in the soluble cytoplasmic fraction. Also like normal liver, a messenger RNA fraction isolated from Morris hepatoma 7800 can be translated by an in vitro protein-synthesizing system into a larger albumin precursor containing L-Ieucine in positions 7, 8, 9, and 10. These studies indicate that Morris hepatomas 5i23C and 7800, while synthesizing smaller amounts of albumin than does normal liver are, however, capable of normal processing of precursor into serum albumin and appear to secrete serum albumin into the incubation medium. 1 Supported by USPHS Grants AM20888 and HLO9O1 1 , and in part by Grant CA 10729. 2Towhomrequestsfor reprintsshouldbeaddressed. Received June 29. 1978; accepted October 3, 1978. Albumin, in normal rat liver, is assembled on polysomes attached to the membranes of the ER3and is transported in stepwise fashion to the lumen of the rough ER, the smooth ER, and the Golgi where it is concentrated and packaged within secretory vesicles. These vesicles then move to the plasmalemma at the hepatic blood front, fuse with it, and empty nascent albumin into the space of Disse (14). During this intracellular pathway, albumin is thought to undergo several modifications. For instance, recent studies indicate that, when albumin mRNA is translated by a cell-free pro tein-synthesizing system, the protein product contains an octadecapeptide extension at the amino terminus (26, 27, 30). This suggests that albumin, like other secretory pro teins (1), is first synthesized as a larger molecule (preproal bumin) with a “signal― hydrophobic extension at its amino terminal end and that during vectorial discharge into the ER this hydrophobic peptide is enzymaticaliy cleaved to yield proalbumin, which then contains a basic hexapeptide extension at the amino terminus (i6, 21). This precursor protein exists in the ER and is converted into albumin in the Goigi cell fractions, just prior to its secretion into the blood (4, 7, i8). The above mechanism for the biosynthesis and intracel lular transport of albumin may, however, be different in some Morris hepatomas, since early studies indicated that Morris hepatomas 5i23C and 9i2i do not secrete albumin into the blood stream of eviscerated rats (23, 24); this idea was substantiated by Uenoyamaand Ono (30),who further indicated that, in contrast to normal liver, Morris hepatoma 5i23C synthesizes albumin on free rather than on mem brane-attached polysomes. Later, McLaughlin and Pitot (i3) showed that, in contrast to normal rat liver, in Morris hepatomas 5i23C and 7800, the nascent albumin peptide chains could be detected antigenically on free polysomes but not on membrane-bound polysomes. These data sup port the hypothesis that these Morris hepatomas, which are thought not to secrete albumin, may contain a deficient mechanism for the attachment of albumin-synthesizing polysomesto the membraneof the rough ER, which there fore precludes the vectorial discharge of albumin into the lumen of the rough ER and its subsequent secretion. Following this rationale, Strauss et al. (26) demonstrated that mRNA isolated from rat hepatoma 5i23C, like that obtained from normal rat liver, directs the in vitro synthesis of preproalbumin; they therefore suggested that, since this tissue is thought not to secrete albumin, some component 3Theabbreviations usedare:ER,endoplasmicreticulum;KRBS,Krebs Ringer-bicarbonate-saline; TCA, trichloroacetic acid; SDS, sodium dodecyl sulfate.