Rapidly labeled nuclear and cytoplasmic renal RNA.

Much of the RNA in mouse kidneys is not recovered when they are extracted with phenol-SDS at 60 degrees C. Better recovery is achieved by extracting in hypertonic buffer with phenol-SDS, chloroform, and isoamyl alcohol after treatment with DNase. 1. Among the polydisperse RNA in the renal nuclei are small amounts of 45S RNA and large amounts of 32S RNA; the latter first appear when 18S RNA is detectable in the cytoplasm 10 min after administration of undine-H(3). 2. In the cytoplasm rapidly labeled 18S RNA is transported as part of a 45S particle to which mRNA is attached. 3. Compared to HeLa cells in culture, normal mouse kidney is poor in the polydisperse nuclear precursors of rRNA and rich in the cytoplasmic polydisperse RNA that sediments like mRNA.

Sequential changes in classes of RNA during compensatory growth of the kidney.

The synthesis of nuclear precursors of rRNA and of cytoplasmic mRNA has been estimated in mouse kidneys following contralateral nephrectomy. 1. The production of nuclear rRNA parallels mitotic activity in the proximal tubule cells. Its peak is 2 days after nephrectomy; there may be a second peak at 8 days. 2. The production of mRNA is almost the reciprocal of rRNA production. It is least at 2 days and most at 4 days after nephrectomy. It may also decrease immediately after nephrectomy. 3. Growing kidney cells may build a store of ribosomes before they elaborate mRNA. The sequential relation between rRNA and mRNA synthesis raises the possibility that production of rRNA could regulate the production of mRNA and that the stimulus to compensatory hypertrophy might need to act only long enough to set into motion the machinery for making rRNA, either directly or as a consequence of exhausting mRNA.

Metabolism of ribosomal precursor ribonucleic acid in kidney.

The labile precursors of ribosomal RNA in mouse kidney are preserved when nuclei rapidly isolated after sieving through multiple screens are swollen and cleansed in the presence of an RNase inhibitor before digestion with DNase and phenol extraction. The kinetics of nucleolar labeling analyzed on polyacrylamide gels show that 36S RNA is the major intermediate product in the catabolism of the original 45S RNA precursor to 32S RNA, from which 28S RNA is derived. Each kidney nucleus contains about 200-600 molecules of 45S RNA; the turnover time of the 45S pool is about 3 +/- 2 min. Compared with HeLa cells, kidney nuclei have a different major intermediate product and a much smaller and more rapidly turning-over pool of ribosomal precursor RNA.

Development of the metanephric kidney. Protein and nucleic acid synthesis.

The metanephric kidney was studied in fetal and older mice beginning at 16 days after mating of the parents. Polyribosomes from fetal kidneys labeled in vitro with 14C-labeled amino acids had 10-20 times more acid-precipitable radioactivity associated with them than polysomes from adult kidneys similarly labeled. Between 3 and 6 days after birth the rate incorporation of labeled amino acids by polyribosomes from neonatal kidneys declined sharply to only twice the value found for adult kidneys. There was no change in the shape of the polyribosome profile with increasing age, but before birth few, if any, ribosomes were bound to membranes compared with 20% 2 days after birth and between 20 and 30% in the adult. Total protein represented less than 10% of the wet weight in the fetal kidney but increased to 17% of the wet weight in the adult kidney. There was a steady decline in the concentration of RNA and DNA with respect to dry weight throughout kidney development. DNA concentration declined more rapidly than RNA concentration, so that the milligram to milligram ratio of RNA to DNA increased. In males the RNA/DNA ratio was stable at 1.3 at 40 days after birth; but in females the decline in DNA concentration was more protracted, and at 200 days after birth the RNA/DNA ratio was only 0.99. Thus, total nucleic acids show only gradual changes in concentration throughout development of the kidney, but a sharp change in the synthetic activity of the ribosomes and in their binding to membranes occurs in kidneys soon after birth.

Conservation of ribosomal RNA during compensatory renal hypertrophy. A major mechanism in RNA accretion.

After removal of one mouse kidney, compensatory hypertrophy in the remaining kidney is marked in 2 days by a 20% average increase in ribosomal RNA (rRNA) per cell. Both 28S and 18S RNA are conserved during the initial stages of compensatory renal hypertrophy to an extent sufficient to account for the rest of the observed accumulation of rRNA. Like some cultured cells, the kidney conserves rRNA during physiological growth.

Membrane-bound ribosomes in kidney: methods of estimation and effect of compensatory renal growth.

Membrane-bound ribosomes are thought to secrete protein for export and free ribosomes to secrete protein for intracellular use. The proportion of the total ribosomes that is bound to membranes in normal mouse kidneys has been estimated by three different methods, and the results have been compared with those obtained by a fourth method used by us previously. The most valid estimates appear to be those obtained (a) by comparison of radioactivity in peaks representing free and membrane-bound ribosomes on linear sucrose gradients after labeling for 24 hr with (14)C-orotic acid, and (b) by measurements of optical density in free and bound ribosomes that had been separated by centrifugation on discontinuous gradients of 0.5 M/2.0 M sucrose. Analyses by these methods show that about 20-25% of the ribosomes in a postnuclear supernatant prepared from mouse kidneys, but only 10-15% of the ribosomes in a post-mitochondrial supernatant, are membrane-bound. About 75% of the bound ribosomes sediment as polysomes of many different sizes. The proportion of membrane-bound ribosomes and their aggregation into polysomes were unchanged in kidneys undergoing compensatory hypertrophy after removal of the opposite kidney. These experiments show that, unlike liver, kidney has a predominance of free ribosomes compared to bound ribosomes; those ribosomes that are membrane-bound do not become free during compensatory renal growth.

Kinetics of RNA synthesis in toad bladder epithelium: action of aldosterone during the latent period.

Processing of RNA in the toad bladder was analyzed by polyacrylamide-gel electrophoresis to determine whether aldosterone causes any changes in the 1 hr before it potentiates transport of sodium ion. No change was found in the quantity or in the specific activity of bulk RNA labeled with uridine-5-(3)H. In vivo and in vitro with either uridine-5-(3)H or with methionine-(methyl)-(3)H as precursors, processing of RNA was extremely slow. Heterodisperse RNA was obvious after 30 min of continuous labeling, but labeling of the 40S precursor of ribosomal RNA was not apparent for 60 min. Labeling of mature 28S and 18S RNA first became apparent after 8 hr. approximately 7S RNA was the principal fastmigrating species labeled at 30 min, and 4S RNA was not heavily labeled until 1 hr. Aldosterone (5 x 10(-7) mole/liter) produced no changes. If care were not taken to inhibit metabolism of native bacteria colonizing the bladder, bacterial RNA of high specific activity predominated. We conclude that RNA metabolism in the toad bladder is extraordinarily slow, that a major acceleration of de novo synthesis in response to physiologic doses of aldosterone was not demonstrable, and that some reports to the contrary may have been influenced by artifacts from bacterial RNA metabolism. Earlier evidence for obligatory alterations in RNA metabolism during the latent period is not strong.

Expression of two "immediate early" genes, Egr-1 and c-fos, in response to renal ischemia and during compensatory renal hypertrophy in mice.

To identify specific genetic regulatory mechanisms associated with renal ischemia, we measured the accumulation of Egr-1 and c-fos mRNAs in the mouse kidney after occlusion of the renal artery and reperfusion. At 1 h after right nephrectomy and arterial occlusion of the contralateral kidney for 10 or 30 min, Egr-1 mRNA levels were three to five times greater in these kidneys as compared with those in control animals that had sustained unilateral nephrectomy alone and were much greater than levels in the normal organ. Whether ischemia was imposed for 10 or for 30 min, renal Egr-1 mRNA contents were equivalent and remained elevated after 24 h of reperfusion subsequent to 30 min of ischemia. Although c-fos mRNA also accumulated in response to ischemia and reperfusion, the pattern differed from that of Egr-1 in that c-fos mRNA content varied with the duration of ischemia and was undetectable 24 h after injury. Contralateral nephrectomy was not necessary to see the marked accumulation of Egr-1 and c-fos mRNAs with unilateral ischemia. Reflow was necessary, however, since only minimal sequence accumulation occurred by the end of the ischemic period. After left uninephrectomy alone, Egr-1 mRNA levels in the remaining kidney were maximal 30 min after surgery, but were not detectable thereafter; c-fos mRNA levels did not change after unilateral nephrectomy. Differential expression of early growth-related genes implicated in transcriptional activation may influence tissue recovery after renal ischemia.

Fibrin deposition in autochthonous Syrian hamster pancreatic adenocarcinomas induced by the chemical carcinogen N-nitroso-bis(2-oxopropyl)amine.

Immunofluorescence studies have demonstrated the presence of fib (a group of fibrinogen- and fibrin-related proteins that react with antibodies raised against fibrinogen) in the stroma of several transplantable animal and autochthonous human tumors. Acceptance of these reports was tempered by the possibility of artifactual clotting and fibrinolysis associated with tumor removal or tumor transplantation and by the relatively poor histology inevitable when immunofluorescence is performed on frozen tissue sections. An immunoperoxidase study therefore was undertaken of the ductal pancreatic carcinomas induced in female LGV Syrian hamsters by N-nitroso-bis(2-oxopropyl)amine [(BOP) CAS: 60599-38-4]. Artifactual clotting and fibrinolysis associated with tumor removal were avoided by systemic anticoagulation and antifibrinolysis. Fibronectin and residual fib were prominent components of tumor stroma. Prominent fib deposits also were found in a new location: the basement membrane zones of atypical pancreatic ducts and invasive carcinomas. In contrast, fib deposits were never found in the basement membranes of blood vessels, nerves, or pancreatic acini of BOP-treated or normal animals, or in the ductal basement membranes in the normal pancreas. Ducts with marked atypicality and invasive pancreatic carcinomas frequently exhibited discontinuous basement membrane staining for fib, which often paralleled loss of staining for the integral basement membrane proteins--type IV collagen and laminin. Loss of acquired fib basement membrane staining with malignant disease progression may serve as a new marker for local tumor invasion.

Augmentation of 1,2-dimethylhydrazine-induced colon cancer by experimental colitis in mice: role of dietary vitamin E.

Because ulcerative colitis predisposes to colonic cancer, for determination of the effect of colitis on experimental colon carcinogenesis, rectal instillations of peptides that attract and activate neutrophils were used to induce colitis in CD-1 (ICR) BR mice receiving 20 weekly injections of the carcinogen 1,2-dimethylhydrazine [(DMH) CAS: 540-73-8]. From week 4 through week 15 of DMH injections, twice-weekly enemas of formyl-norleucyl-leucyl-phenylalanine were given to DMH-treated mice. The effect of the antioxidant vitamin E in the diet (1,750 IU/kg diet) was studied in another group of mice treated with DMH and having colitis. Four weeks after DMH was discontinued, cancer occurred in 9 of 28 (32%) animals with DMH plus control enemas, in 22 of 29 (76%) animals with DMH plus colitis (P = .001), and in 16 of 28 (57%) animals with DMH plus colitis plus supplemental vitamin E (P = .11 compared with the group with DMH and colitis). Colitis enhances DMH-induced colonic carcinogenesis.

Pancreatic cancer in the Syrian hamster induced by N-nitrosobis(2-oxopropyl)-amine: cocarcinogenic effect of epidermal growth factor.

Because epidermal growth factor (EGF) is rapidly bound and internalized into rat pancreas, stimulates uptake of tritiated thymidine, and increases pancreatic weight, a cocarcinogenic effect on pancreatic cancer seemed likely. Pancreatic adenocarcinomas were induced in 70 female Syrian hamsters by 19 weekly s.c. injections of N-nitrosobis(2-oxopropyl)amine (BOP) (10 mg/kg). From Wk 5 through Wk 8 of BOP injections, additional s.c. injections of EGF (5 micrograms every 3 days for 10 injections) were given to 45 animals, while 25 received saline solution. An additional group of 10 received EGF alone, and another 10 animals received saline solution alone (controls). Eleven wk later, the mean body weight of EGF-treated animals increased by 29% as compared with that of controls, and their mean pancreatic weight relative to body weight increased by 44% as compared with controls. The mean body weight of EGF + BOP-treated animals increased by 10%, and their pancreatic weight relative to body weight increased by 22% as compared with that of animals treated with BOP alone. The incidence of pancreatic cancer in the EGF + BOP-treated animals was 75% versus 44% in those treated with BOP alone (P = 0.016). No tumors developed in either animals treated with EGF alone or control animals. EGF augments pancreatic carcinogenesis induced by BOP. The incidence of bronchial carcinomas doubles.

Effects of alpha-difluoromethylornithine and 5-fluorouracil on the proliferation of a human colon adenocarcinoma cell line.

Because alpha-difluoromethylornithine (DFMO) reduces the incidence of experimental colon cancers, inhibits the growth of human lung cancer cells and human leukemia cells in culture, and in combination with methylglyoxal (bis)guanylhydrazone induces remission in children with leukemia, its effectiveness against a human colon adenocarcinoma cell line (Colo 205) was tested alone and in combination with 5-fluorouracil (5-FU). Both DFMO (2 X 10(-4) M) and 5-FU (10(-6) M) inhibited Colo 205 cell proliferation. Above 5 X 10(-4) M DFMO (p less than 0.001) and at 10(-4) M 5-FU (p less than 0.001), Colo 205 growth was completely inhibited. Although DFMO did not sensitize Colo 205 cells to a noninhibitory concentration of 5-FU, the effectiveness of inhibitory concentrations of 5-FU and DFMO in reducing Colo 205 cell growth was additive. DFMO (2 X 10(-4) M) caused 89 to 93% inhibition of ornithine decarboxylase activity (p less than 0.001) and reduced levels of putrescine (93%; p less than 0.01) and spermidine (57%; p less than 0.02). Growth rate and the intracellular putrescine and spermidine contents were restored by 10(-6) M putrescine. DFMO could be an effective chemotherapeutic agent against human colonic cancer because of its effects at such unusually low concentrations in vitro.

Effects of alpha-difluoromethylornithine on the growth of experimental Wilms' tumor and renal adenocarcinoma.

Because polyamines are essential for cellular growth and differentiation, and because human renal carcinomas have spermidine levels that are higher than those in normal renal tissue, effects of 2-difluoromethylornithine (DFMO) on the growth of experimental renal tumors were investigated. DFMO is a specific enzyme-activated irreversible inhibitor of ornithine decarboxylase, the rate-limiting enzyme controlling polyamine biosynthesis. DFMO (2%) in drinking water was administered to BALB/c mice with intrarenal transplants of a renal adenocarcinoma cell suspension and to Wistar/Furth rats with s.c. transplants of a Wilms' tumor. At 28 days, renal carcinomas in DFMO-fed mice weighed 72% less than those in control animals (p less than 0.001). Wilms' tumor weight was not affected by DFMO feeding. DFMO caused 72 to 75% inactivation of ornithine decarboxylase activity and reduced putrescine levels in renal carcinoma and Wilms' tumor, reduced spermidine levels in Wilms' tumor, and apparently raised spermine levels in the latter as a consequence. DNA content was not affected by DFMO feeding. The mean number of lung metastases in DFMO-fed, renal carcinoma-bearing mice was 0.1 and in controls was 1.4 (p less than 0.001). DFMO feeding increased survival of mice bearing renal carcinomas by 3.0 +/- 0.8 (S.E.) days (p less than 0.05), i.e., from 30.5 +/- 0.8 days to 33.5 +/- 1.2 days. DFMO did not affect the growth of Wilms' tumor; however, in renal adenocarcinoma, it reduced growth, prevented lung metastases, and increased survival.

Contrasting effects of subtotal enteric bypass, enterectomy, and colectomy on azoxymethane-induced intestinal carcinogenesis.

Compensatory hyperplasia after extensive loss of functioning small or large intestine might predispose to the development of neoplasia in the residual adapted bowel. To test this hypothesis, male Fischer rats were randomized to receive 85 to 90% jejunoileal resection or bypass, subtotal colectomy, or no operation (controls). One week later, the first of six weekly s.c. injections of azoxymethane (15 mg/kg/week) was given. At the 36th week postoperatively, mean body weight after enterectomy or colectomy it was 78 to 79% of control. Adaptation after all three operations was characterized by 22 to 84% increments in villous height and crypt depth in the residual functioning ileum (p = 0.05 to 0.001); the depth of colonic crypts was unchanged. Fewer rats developed intestinal tumors after enteric bypass (36%) than after any of the other treatments (80 to 91%) (p = 0.01 to 0.001); the depth of colonic crypts was unchanged. Fewer rats developed intestinal tumors after enteric bypass (36%) than after any of the other treatments (80 to 91%) (p = 0.01 to 0.001). Compared with controls, bypass reduced the number of colonic tumors by 77% (p less than 0.001). Although resection did not affect colonic tumor yield, it tripled the incidence of tumors in the duodenum and jejunum (p = 0.025). Colectomy promoted rectal carcinogenesis (p less than 0.05). Anastomotic tumors were commoner after intestinal resection. the lower frequency of tumors after jejunoileal bypass contrasts with enhanced carcinogenesis after enterectomy or colectomy. Profound reduction in body weight may prevent the promotional effect of adaptive hyperplasia.

Promotion of azoxymethane-induced colonic neoplasia by resection of the proximal small bowel.

Potential enhancement of intestinal neoplasia by compensatory mucosal hyperplasia was tested in rats subjected to 50% proximal small bowel resection (PSBR) 10 days after the last of 16 weekly injections of azoxymethane. Azoxymethane alone increased jejunal contents of RNA and DNA each by 26% at 17 to 18 weeks (p less than 0.01) before there was macroscopic evidence of neoplasia. Three months after PSBR alone, ileal hyperplasia was characterized by increased amounts of RNA (42 to 76%) and DNA (68 to 95%), taller villi, deeper crypts, and luminal dilation (p less than 0.05 to 0.001); however, the colon showed only patchy hyperplasia. When the combined effects of azoxymethane and PSBR were observed 26 to 30 weeks after the first injection, rats with PSBR had an increased number of colonic tumors per animal (2.9 versus 1.6 for controls; p less than 0.02). Despite the intense ileal hyperplasia produced by PSBR, ileal neoplasia did not occur. Enhanced colonic carcinogenesis followed sequential exposure of the mucosa to the carcinogen (azoxymethane) and to the promoting factor (PSBR).

Inhibition of ornithine decarboxylase with 2-difluoromethylornithine: reduced incidence of dimethylhydrazine-induced colon tumors in mice.

2-Difluoromethylornithine (DFMO) was administered to 1,2-dimethylhydrazine (DMH)-treated mice to reduce colonic polyamine levels and mucosal hyperplasia. Mice received 1% DFMO in drinking water throughout the experiment and were given injections of DMH (20 mg/kg) weekly for 28 weeks. DFMO inactivated 93% of colonic ornithine decarboxylase activity. Although DMH treatment did not induce colonic ornithine decarboxylase activity by Week 28, the putrescine content was increased 31% in DMH-treated mice (p less than 0.01). Concurrent treatment with DFMO depressed putrescine content (42 to 63%) and spermidine content (27 to 38%), but it increased spermine content (18 to 22%). At Week 28 of treatment with DMH alone, RNA content was increased 8.6% (p less than 0.01), DNA content 10% (p less than 0.01), DNA specific activity 24% (p less than 0.01), and crypt depth 20% (p less than 0.01), but not in mice receiving DMH and DFMO. At 28 weeks, 13 of 17 mice (76%) treated with DMH alone had histologically confirmed colon cancers; of mice treated with DMH and DFMO, two of 18 (11%) had colonic tumors. Throughout the experiment, 50 colon cancers developed in 16 DMH-treated mice (mean, 3.12 tumors/mouse); three mice treated with DMH and DFMO developed three colon cancers total (p less than 0.001). Reduction of colonic polyamine levels after DFMO treatment prevents proliferative changes induced by DMH and reduces the incidence of tumors.

Physical aspects and cytoplasmic distribution of messenger RNA in mouse kidney.

As a prerequisite to examining mRNA metabolism in compensatory renal hypertrophy, polyadenylated RNA has been purified from normal mouse kidney polysomal RNA by selection on oligo(dT)-cellulose. Poly(A)-containing RNA dissociated from polysomes by treatment with 10 mM EDTA and sedimented heterogeneously in dodecyl sulfate-containing sucrose density gradients with a mean sedimentation coefficient of 20 S. Poly(A) derived from this RNA migrated at the rate of 6-7 S RNA in dodecyl sulfate-containing 10% polyacrylamide gels. Coelectrophoresis of poly(A) labeled for 90 min with poly(A) labeled for 24 h indicated the long-term labeled poly(A) migrated faster than pulse-labeled material. Twenty percent of the cytoplasmic poly(A)-containing mRNA was not associated with the polysomes, but sedimented in the 40-80 S region (post-polysomal). Messenger RNA from the post-polysomal region had sedimentation properties similar to those of mRNA prepared from polysomes indicating post-polysomal mRNA was not degraded polysomal mRNA. Preliminary labeling experiments indicated a rapid equilibration of radioactivity between the polysomal and post-polysomal mRNA populations, suggesting the post-polysomal mRNA may consist of mRNA in transit to the polysomes.

Prognosis of clinical stage I melanoma patients with positive elective regional node dissection.

We tested 12 clinical and histologic variables to see which ones best predicted death from melanoma in 66 patients with positive elective regional node dissections (clinical stage I, pathologic stage II [CSI, PSII]). Despite the presence of lymph node metastases, not all patients had poor prognoses. Patients with tumors less than or equal to 3.5 mm and a percentage of positive nodes less than or equal to 20% had a 7-year survival rate of 66%. Within this low-risk group the subset with primary lesions on the trunk or extremities (except hands and feet) had a 7-year survival rate of 76%. This compares with poor 7-year survivals of 29% and 30% observed in other defined high-risk groups. Our results confirm and extend earlier observations concerning the prognoses of CSI, PSII melanoma patients and are relevant to any ongoing and future studies concerning elective regional node dissection (ERND) or adjuvant therapy trials in melanoma.

Compensatory renal hypertrophy in the absence of androgen binding.

As a direct test for a role of androgens, compensatory renal hypertrophy was studied in normal male mice, in androgen-insensitive Tfm/Y mice, and in sibling normal female mice. Fifteen days after unilateral nephrectomy, although the kidneys of the normal male mice were larger, relative increases in renal weight were similar in all groups (33-43%). The magnitude of the increase and the contents of protein, RNA, and DNA were the same in the Tfm/Y mice and the female mice. Androgens are not essential to compensatory renal hypertrophy, but they promote larger mice with larger kidneys.

Renal angiomyolipoma with sarcomatous transformation and pulmonary metastases.

We report the case of a 49-year-old woman with a large renal angiomyolipoma that invaded the liver. In some areas, the tumor had the appearance of a typical angiomyolipoma; however, it also had foci where the spindle cells of the lesion showed marked cytologic atypia and mitotic activity, giving it the appearance of a high-grade sarcoma. Immunohistochemical studies demonstrated expression of vimentin, desmin, and muscle-specific actin by the sarcoma cells; these findings were consistent with leiomyosarcoma. A second, small typical angiomyolipoma was also present in the kidney. In addition, the liver exhibited focal nodular hyperplasia. Three weeks after resection of the primary renal tumor, pulmonary metastases were diagnosed by fine-needle aspiration biopsy. This is the first report of a case of angiomyolipoma with sarcomatous transformation and biopsy-proven metastatic disease.