We constructed transgenic mice containing a renin-promoter SV40 T antigen fusion transgene with the intention of inducing neoplasia in renin-expressing cells and isolating renin-expressing cell lines in vitro. We examined six kidney tumors from mice representing three different transgenic lines and found they expressed their endogenous renin gene. Initially, five nonclonal kidney tumor-derived cell lines were established which expressed their endogenous renin gene in addition to the transgene. They retained active renin intracellularly and constitutively secreted an inactive form of renin (prorenin). One of these cell lines was cloned to homogeneity. This line maintained high level expression of renin mRNA throughout 3 months of continuous culture. Although the cells contained an equal proportion of active and inactive renin, the species constitutively secreted into the media was predominantly (95%) prorenin. However, active renin secretion was stimulated 2.3- and 4.6-fold by treatment with 8-bromo-cAMP after 4 and 15 h, respectively. In addition, the presence of multiple secretory granules was confirmed by ultrastructural analysis. These cells, which express renin mRNA and can regulate secretion of active renin, should provide an excellent tool for studying renin gene regulation and secretion. Furthermore, these mice should provide a useful source for the establishment of renin-expressing cell lines from a variety of renin-expressing tissues.
Summary A subline of mouse Sarcoma 180 (S-180) cells, resistant to adenosine analogs, was selected in cell culture by serial passage in the presence of increasing concentrations of N 6 -furfuryladenosine. These cells (S-180/KR) were cross-resistant to a number of other adenosine analogs, including N 6 -(Δ 2 -isopentenyl)adenosine (IPAR). The resistance was unaltered after 5 months maintenance in absence of the drug. S-180/KR cells showed increased sensitivity to certain purine and pyrimidine nucleoside analogs such as 6-thiopurine ribonucleoside and 5-fluorodeoxyuridine. The activity of purine nucleoside phosphorylase (purine nucleoside:orthophosphate ribosyltransferase, EC 2.4.2.1) an enzyme that in crude cell extracts but not in whole cells cleaved IPAR-8- 14 C to the noncytotoxic free base at 10% the rate for inosine, was equal in sensitive and resistant cells. In contrast, the activity of adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4), an enzyme that provides an alternate metabolic pathway for utilization of adenosine (K m 29 µm) was increased 45% in extracts of resistant cells. No cleavage of IPAR by this enzyme to inosine in cells or cell extracts was observed, but IPAR was a competitive inhibitor of adenosine deamination with a K i of 1.3 mm. The change that appears to be solely responsible for resistance to adenosine analogs was the 20,000-fold reduction in the activity of adenosine kinase (adenosine 5′-triphosphate:adenosine 5′-phosphotransferase, EC 2.7.1.20). This enzyme converts adenosine and its analogs to their 5′-monophosphates. The elution pattern of adenosine kinase of both cells on diethylaminoethyl cellulose and K m for adenosine (0.5 µm) were identical. In resistant cells, deficient in adenosine kinase but containing increased adenosine deaminase, the rate of uptake of adenosine was slightly reduced; V max 18.2 and 5.1 nmoles/hr/mg cells in S-180 (K t 47 µm) and S-180/KR (K t 20 µm), respectively. These changes were reflected in altered growth response to adenosine when de novo synthesis of purine nucleotides was blocked by amethopterin. In sensitive, but not in resistant cells, IPAR accumulated in trichloroacetic acid-soluble pool in the form of 5′-monophosphate, which comprised 75% of cellular IPAR metabolites. In resistant cells deficient in adenosine kinase and unable to otherwise metabolize IPAR, the rate of uptake of IPAR was greatly reduced; V max 3.3 and 0.08 nmoles/hr/mg cells in S-180 (K t 24 µm) and S-180/KR (K t 14 µm), respectively.
Liver tumors from interspecific hybrid, transgenic mice containing the SV40 early region linked to a mouse major urinary protein enhancer/promoter were analyzed for loss of heterozygosity to identify chromosomal regions which potentially contain genetic loci involved in multistep tumorigenesis. A broad pattern of complete and partial loss of heterozygosity or allelic imbalance was observed with frequent loss of heterozygosity/partial loss of heterozygosity of loci on chromosomes 1, 5, 7, 8, and 12. In tumors from Mus domesticus x Mus spretus F1 mice a strong preference for loss of the domesticus allele of H19 on chromosome 7 was observed, whereas loss of heterozygosity/partial loss of heterozygosity on chromosome 8 involved preferential loss of spretus alleles. In tumors from reciprocal crosses with Mus castaneus, the maternal chromosome 7 H19 allele was preferentially lost irrespective of whether it was domesticus or castaneus, strongly suggesting the involvement of an imprinted gene(s) in tumor progression.
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