Steven R. Sylvester

Increased expression of c-myc and c-H-ras in dichloroacetate and trichloroacetate-induced liver tumors in B6C3F1 mice

The expression of c-myc and c-H-ras in hyperplastic nodules and hepatocellular carcinomas induced in male B6C3F1 mice after chronic administration of dichloroacetate (DCA) and trichloroacetate (TCA) was studied using in situ hybridization. Expression of c-myc and c-H-ras mRNA was increased in both nodules and carcinomas relative to surrounding tissue and tissues obtained from control animals. Myc expression was similar in hyperplastic nodules and carcinomas induced by DCA, but was significantly higher in TCA-induced carcinomas than in hyperplastic nodules and carcinomas produced by DCA. In carcinomas from animals whose TCA treatment was suspended at 37 weeks, c-myc expression remained high relative to control and surrounding liver tissue at 52 weeks. In contrast, the expression of c-H-ras was consistently elevated in carcinomas from both treatments relative to hyperplastic nodules and non-tumor tissue. Within carcinomas from both treatments, focal areas could be located which expressed even higher levels of c-myc. This heterogeneity was not observed in carcinomas hybridized to c-H-ras-probes. These data suggest that elevated expression of c-H-ras and c-myc might play an important role in the development of hepatic tumors in B6C3F1 mice. Elevated expression of c-H-ras was closely associated with malignancy. Increased c-myc expression does not seem necessary for progression to the malignant state. On the other hand, the increased expression of c-myc appears related to the earlier progression of TCA-induced tumors to the malignant state.

Sequence and Post‐translational Modification of Sulfated Glycoprotein‐2 Secreted from Rat Sertoli Cellsa

Sulfated glycoprotein-2 (SGP-2) is the most abundant of the six or seven major proteins secreted by cultured rat Sertoli cells. SGP-2 is secreted from Sertoli cells as a disulfide-linked heterodimer whose reduced subunits migrate with mobilities of 47 kDa and 34 kDa in SDS-polyacrylamide gels. SGP-2 contains N-linked carbohydrate (23.7% of total MW), and the sulfate moiety is associated entirely with the glycosylated portion of the protein. Indirect immunofluorescence indicates that after secretion, SGP-2 becomes associated with late spermatids and appears to concentrate on the acrosome, neck, and distal tail portions of mature spermatozoa. In addition, SGP-2 is also synthesized by the epididymis. Pulse-chase studies with [35S]methionine were used to examine SGP-2 biosynthesis in cultured rat Sertoli cells. SGP-2 is synthesized as a cotranslationally glycosylated 64 kDa precurser with a singular PI of 7.5. The SGP-2 precursot is post-translationally modified to a heterogeneously charged 73 kDa protein with an average PI of 4.5. The modified precursor is then proteolytically cleaved to the mature 47 kDa and 34 kDa subunits before secretion to the extracellular space. We have previously shown that the acidic PI and charge heterogeneity of SGP-2 is due, at least in part, to sialation and sulfation of carbohydrate. We conclude that these carbohydrate modifications occur prior to proteolytic processing and secretion of SGP-2. A full-length cDNA clone for SGP-2 was isolated, and the nucleotide sequence was determined. The 1857 nucleotide-long cDNA consists of a 297 nucleotide 5’ noncoding segment, a coding segment of 1341 nucleotides, and 219 nucleotides of 3’ noncoding sequence. The encoded protein has a molecular mass of 51,379 daltons and contains 447 amino acids. The large and small subunits of mature SGP-2 were isolated, and the partial amino acid sequence of each N-terminus was determined by gas phase sequencing. These data allowed assignment of Gly-21 as the site of leader peptide cleavage and Arg-226 as the site for proteolytic processing of pro-SGP-2. The small subunit of SGP-2 contains two potential N-glycosylation sites (Asn-X-Ser or Asn-X-Thr), whereas the large subunit contains four. A molecular weight difference of 14 kDa is observed between the glycosylated and deglycosylated SGP-2 precursor, suggesting that all six N-glycosylation sites are occupied. Five cysteine residues are available in each subunit for the formation of disulfide bonds.