Neurophysin biosynthesis in vitro in oat cell carcinoma of the lung with ectopic vasopressin production.

The incorporation of labeled compounds into neurophysins of a transplantable human oat cell carcinoma of the lung with ectopic vasopressin production was studied in vitro. Neurophysins in cell extracts and in incubation media were isolated by immunoprecipitation and analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. When cells were incubated with L-[35S]cysteine for 12 h, SDS-polyacrylamide gel electrophoresis of the immunoprecipitates from cell extract and medium resolved two forms of neurophysins with apparent molecular mass of 10,000 (10K) and 20,000 (20K). Both forms of [35S]-neurophysins were completely displaced from the immunoprecipitates by excess human neurophysin. Incubation of cells with L-[35S]cysteine and D-[3H]-glucosamine hydrochloride revealed that glucosamine was incorporated into the 20K neurophysin region, but not into 10K species. To observe the kinetics of labeling of the two forms of neurophysins, cells were incubated with L[35S]cysteine for varying periods of time. After short labeling periods, most of the radioactivity resided in 20K species, which plateaued after 1 h, whereas 10K neurophysin progressively increased in its height. When cells were chased with unlabeled cysteine after the exposure to a short pulse of labeling, 20K neurophysin peak gradually decreased with an apparent initial half-life of 1 h. In contrast, the label in 10K neurophysin steadily increased, which exceeded the former by 3 h of chase. Analysis of 20K neurophysin in cell extract by isoelectric focusing on polyacrylamide gel demonstrated that it was principally composed of a protein with an apparent isoelectric point (pI) of 5.7. These results suggest that neurophysin is synthesized in ectopic vasopressin-producing tumors by post-translational processing from a glycosylated proneurophysin with an apparent molecular mass of 20,000 daltons and a pI of 5.7.

Production of bone-resorbing activity and colony-stimulating activity in vivo and in vitro by a human squamous cell carcinoma associated with hypercalcemia and leukocytosis.

A squamous cell carcinoma of 33-yr-old patient who developed marked leukocytosis and hypercalcemia was transplanted into nude mice in which more marked leukocytosis and hypercalcemia also developed. This tumor (LJC-1-JCK) produced a colony-stimulating factor (CSF) and formed a cyst in the tumor from which a CSF-producing cell line (T3M-1) was established. The CSF causes predominantly formation of granulocytic colonies in addition to macrophage colonies. Bone-resorbing activity (BRA) was detected in the cystic fluid and was eluted as two separate peaks with proteins of an apparent molecular weight of 30,000-50,000 and 10,000-20,000. Colony-stimulating activity (CSA) was eluted at an apparent 30,000 mol wt. The conditioned medium of the T3M-1 cells also contained a BRA with an apparent 14,000 mol wt, whereas CSA eluted at an apparent 30,000 mol wt. PTH, epidermal growth factor, transforming growth factor-alpha, prostaglandin Es, and vitamin D could not account for the powerful BRA. In contrast to CSA, BRA was not inactivated by trypsin and more stable at 70 degrees C. When T3M-1 cells were transplanted into nude mice, marked hypercalcemia developed in addition to granulocytosis. Our findings suggest that the tumor produces and secretes a powerful BRA in vivo and in vitro, which is different from CSA in terms of molecular weight, heat stability, and trypsin treatment. We speculate that the synergistic action of CSF that stimulates macrophage colony formation and recruits osteoclast precursors, and BRA, which stimulates mononuclear phagocytes and/or osteoclasts were responsible for a marked increase in osteoclastic bone resorption and humoral hypercalcemia in the patient.

Ascites form of a human cancer serially transplantable in nude mice.

Human adenocarcinoma cells injected into the peritoneal cavities of BALB/c nude mice (nu/nu) induced ascites carcinoma. The inoculant was obtained from subcutaneous tumors produced in nude mice by an injection of ascites cells from a patient with carcinomatous peritonitis caused by mucinous adenocarcinoma of the stomach. An ascitic fluid began to accumulate 45 days after inoculation and reached the maximum volume within 120 days. Dispersed stomach cancer cells in the ascites could be serially transplanted in nude mice in an ascites form. The morphology of these cells was similar to that of the original cells in the ascitic fluid of a patient with carcinomatous peritonitis.

Human gastric choriocarcinoma serially transplanted in nude mice.

A gastric choriocarcinoma cell line capable of producing human chorionic gonadotropin (HCG) was studied by sc transplantation and serial passages into nude mice fed under specific pathogen-free (SPF) conditions. The tumor-take rate at the serial heterotransplantation in SPF mice was high (greater than 95%), in contrast with a low rate in coventional animals (35%). The restoration of morphology and function of the original neoplasm were exclusively verified in SPF mice. Multiple lung metastases were found in 2 animals. The production, storage, and excretion of HCG by tumor cells were confirmed by its high content in serum and cystic fluid in tumors and by its intracellular localization. The tumor cells also contained a specific placental alkaline phosphatase in their membranes. The cells were various trophoblastic types ranging from primitive cytotrophoblasts to typical syncytial cells. The hormone effects of the tumor on sex organs of tumor-bearing animals were evident.

Production of human alpha 1-antichymotrypsin-like protein by a human malignant melanoma transplanted into nude mice.

A tissue culture line of a human malignant melanoma, SEKI, induced cachexia in nude mice (BALB/c-nu/nu) (Kondo et al., Cancer Res., 41: 2912-2916, 1981). During the investigation of the cause of the cachexia, the melanoma was found to produce a protein immunologically identical to human alpha 1-antichymotrypsin (alpha 1-Ach). Tissues of the SEKI melanoma contained the protein immunologically equivalent to 0.29 +/- 0.11 (S.D.) mg of human alpha 1-Ach per g of wet tissue, while the other six human malignant tumors transplanted into nude mice did not contain a detectable amount of it. In the serum of nude mice bearing the melanoma, this protein appeared soon after the tumor growth occurred and gradually increased up to the level equivalent to 5 mg of human alpha 1-Ach per dl. Removal of the tumor resulted in a rapid decrease of the protein in the serum to an undetectable level within 1 day. This problem was never detected in the serum of nude mice bearing the other 27 human malignant tumors or controls. Purification of this protein was carried out by the column chromatography using DE-52, Blue-Sepharose, and SP-Sephadex. The elution patterns were the same as those of alpha 1-Ach in human serum, and the molecular weight of the protein was estimated as 69,000 by Sephadex G-100 column chromatography and 65,000 by polyacrylamide gel electrophoresis with sodium dodecyl sulfate. This purified protein, however, did not exhibit inhibitory activity against chymotrypsin. These results show that this melanoma produced a protein immunologically identical and physicochemically very similar to human alpha 1-Ach. This melanoma-nude mouse system may provide a useful model for investigating the synthesis of human alpha 1-Ach and analysis of its physiological roles.

A new simple enzymatic assay method for urinary polyamines in humans.

We developed a new simple enzymatic assay method for measuring urinary polyamines (total amount of putrescine, spermidine, and cadaverine), using an acylpolyamine amidohydrolase and a putrescine oxidase. First conjugated polyamines (putrescine, spermidine, and cadaverine) in urine were hydrolyzed by incubation with an acylpolyamine amidohydrolase at 30 degrees for 1 hr. then, free polyamines were separated by cation-exchange chromatography and incubated with a putrescine oxidase at 30 degrees for 30 min. Hydrogen peroxide formed in this reaction was measured spectrophotometrically (at 514 nm). Polyamine levels in urine were determined in 70 normal subjects, 124 patients with cancer, and 52 patients with diseases other than cancer. Elevation above 3 S.D.s of the normal mean was found in 90 (72.6%) of the 124 patients with cancer and in 6 (11.5%) of the 52 patients with diseases other than cancer. Serial studies in 19 patients with cancer indicated that polyamines in urine were reduced after successful surgery. Our new method is simple and rapid and therefore very useful for routine clinical application. Moreover, our data indicate that the determination of polyamine levels is useful as a marker of disease activity in patients with cancer.

Serum sialyltransferase and liver catalase activity in cachectic nude mice bearing a human malignant melanoma.

Cachexia is rare in nude mice bearing human malignant tumors even when the transplanted tumors become as large as the body size of the host. In our series on heterotransplantation of a variety of human malignant tumors into nude mice, a malignant melanoma (SEKI) was found to induce severe body weight loss in the host at the early stage of transplantation. There was no electrolyte disturbance, hyper- or hypoadrenocorticism, hyperthyroidism, or destruction of cells of vital organs to account for the weight loss. Moreover, no evidence was obtained for concomitant infection with bacteria, Mycoplasma or fungi. These cachectic mice revealed remarkably increased levels of serum sialyltransferase and decreased liver catalase activity. The removal of tumor tissues from these mice resulted in prompt recovery of body weight, serum sialyltransferase, and liver catalase activity within 1 to 2 weeks. On the basis of the results obtained, the SEKI melanoma was thought to have produced a pathophysiological state in host nude mice which was very similar to that of cachexia in cancer patients. Nude mice bearing transplants of SEKI melanoma may provide a useful system for the study of cancer cachexia in humans.

Amplification of growth inhibition by glucocorticoid on L5178Y and L1210 lymphoblasts in vivo.

One component of a factor in Proteus mirabilis (Factor 1) which specifically amplifies the induction of several liver enzymes by glucocorticoid in target cells also increases the growth inhibition of glucocorticoid on the ascitic form of L1210 cells and solid tumors of L5178Y lymphoblasts in vivo. The growth of L5U78Y and L1210 lymphoblasts was inhibited by a triamcinolone acetonide dose of over 0.5 to 1.0 mg/kg body weight. Factor 1 increased the inhibitory effect of a triamcinolone acetonide dose of less than 4.0 mg/kg bodyweight but had little effect on the effects of doses of over 4.0 mg. Factor 1 (10 biological units/kg body weight) itself also caused marked inhibition of the growth of these lymphoblasts without affecting the body weight or adrenal gland weight, its effect being equivalent to that of 3- to 4-mg/kg body weight doses of triamcinolone acetonide alone. There was no significant difference in the level of plasma total corticoids or that of plasma adrenocorticotropic hormone between rats treated with 0.9% NaCl solution or those treated with Factor 1, and Factor 1 had no cytotoxic effec on cultured L5178Y lymphoblasts. Thus, Factor 1 may amplify the effect of physiological level of glucocorticoid in mice sufficiently to inhibit the growth of these lymphoblasts without causing any significant side effects to the host animal.

Immune elimination of host fibroblasts for the cultivation of human tumors transplanted into nude mice.

We report here a useful method for the isolation and cultivation of human tumor cells in vitro from human tumors grown in nude mice. A rabbit was immunized with spleen cells obtained from adult nude mice. The rabbit antiserum in the presence of complement effectively killed cultured cells derived from various mouse tissues, but it was not cytotoxic to cultured cells from human tissues including tumors. When mixed cultures consisting of human tumor cells and nude mouse fibroblasts were treated with the antiserum and complement, the nude mouse fibroblasts were completely removed from the cultures, and the human tumor cells could be propagated without noticeable changes in morphological features. Primary cultures of heterotransplanted human tumors grown in nude mice were also successfully treated, resulting in the ultimate elimination of fibroblastic cells derived from the stroma of the tumor. The functional properties of the tumor cells (production of human chorionic gonadotropin by choriocarcinoma cells and production of carcinoembryonic antigens by pancreas carcinoma cells) were also maintained after the antiserum treatment.

Growth-inhibitory action of an estrogen-chlorambucil conjugate (KM2210) in human breast cancer cell line MCF-7: its relation to reduction of estrogen receptor and transforming growth factor-alpha secretion.

We investigated the effects of a benzoate of an estradiol-chlorambucil conjugate (KM2210) and chlorambucil on growth, estrogen receptor, and secretion of transforming growth factor (TGF)-alpha in the hormone-dependent human breast cancer cell line MCF-7. In the presence of 10(-10)-10(-6) M KM2210, the estrogen-induced growth of MCF-7 was completely inhibited. Inhibited growth of MCF-7 treated with 10(-8) or 10(-6) M KM2210 for 4 days was not rescued by removal of the drug and the addition of estradiol. By treatment of MCF-7 with KM2210 for 4 days, estrogen receptor-binding sites were decreased at 10(-8) M and were not detected at 10(-6) M but were unaltered by 10(-8) M chlorambucil. Moreover, estrogen receptor immunoreactivity and the level of estrogen receptor mRNA were decreased through treatment with 10(-6) M KM2210 for 4 days. These suppressions occurred prior to the onset of inhibitory action on MCF-7 growth. Secretion of TGF-alpha from MCF-7 was decreased by 4 days of treatment with 10(-8) and 10(-6) M KM2210 but not with chlorambucil. The addition of exogenous TGF-alpha generally restored the growth of MCF-7 treated with 10(-8) M KM2210. We concluded that KM2210 has irreversible or at least long-standing inhibitory effect on estrogen-dependent growth of MCF-7. It is conceivable that the decrease of estrogen receptor renders the cell unable to respond to estrogen with increased TGF-alpha secretion and succeeding cell growth.

Establishment and characterization of a human cancer cell line that produces human colony-stimulating factor.

A human colony-stimulating factor (CSF) producing cell line, T3M-1, has been established from explant cultures of a human squamous cell carcinoma of the oral cavity that secretes human CSF. It has been continously propagated during the past 15 months. The cells grew in a monolayered sheet with about 17 hr of population-doubling time and showed a colony-forming capacity with about 5% plating efficiency. The cells exhibited an epithelioid morphology resembling the structure of the original tumor, and they showed "tumor takes" when inoculated into nude mice. Karyotypic analysis revealed the cell line to be a human aneuploid one with a hypotriploid mode, including the Y-chromosome(s) and at least 10 common markers. T3M-1 cells possess the characteristic function of human CSF production in vitro, and a marked neutrophilia was observed in nude mice bearing the tumors produced by inoculation with the T3M-1 represents a new human cell line that secretes human CSF.

Inappropriate secretion of antidiuretic hormone in nude mice bearing a human bronchogenic oat cell carcinoma.

A 58-year-old man with bronchogenic oat cell carcinoma developed a typical syndrome of inappropriate secretion of antidiuretic hormone. The tumor tissue obtained at autopsy had been serially transplanted in nude mice for more than four years with 20 passages. The levels of vasopressin were remarkably increased in the plasma of nude mice bearing this tumor [24.4 +/- 18.3 (S.D.) pg/ml, n = 3] as well as in the tumor tissues ]134.3 +/- 72.2 ng/g, n = 3]. Furthermore, human nicotine-stimulated neurophysin was detected in both plasma and tumor tissues (7.4 +/- 3.7 ng/ml, n = 3, and 2.28 +/- 0.90 micrograms/g, n = 3, respectively). On ad libitum intake of water, nude mice bearing this tumor excreted significantly less urine with higher sodium concentration than did controls, but serum sodium concentrations did not differ from those of controls. When tumor-bearing mice were hydrated with 2 ml of water twice a day i.p., their diuretic response was found to be suppressed in parallel with the tumor size. However, these mice did not become hyponatremic because they drank less water. When a larger amount of water was loaded which could not be compensated by restriction of water drinking, serum sodium concentrations were markedly decreased. On the basis of these results, the lung cancer, when transplanted into nude mice, produced and secreted its own antidiuretic hormone, which induced inappropriate secretion of antidiuretic hormone in the mice. These mice may provide a useful experimental model for the study of excessive secretion of antidiuretic hormone and associated pathophysiological disorders.

Association of hypercalcemia with tumors producing colony-stimulating factor(s).

Two human malignant tumors, which we previously reported to produce colony-stimulating factors (CSFs), were found to be accompanied by remarkable hypercalcemia. A patient with a CSF-producing lower jaw cancer (squamous cell carcinoma) developed a marked granulocytosis (150,000/microliters) and hypercalcemia (more than 215 mg/dl). The tumor was successfully transplanted into nude mice, which developed marked granulocytosis (300,000/microliters) and hypercalcemia (20 mg/dl). White blood cell and serum calcium concentrations of these mice decreased promptly to normal levels when the tumor was excised. Treatment with prednisolone (1.5 mg/kg) or indomethacin (5 mg/kg) had no effect on the serum calcium level of these mice. Parathyroid hormone or prostaglandin E was not increased in the serum of the mice or in the tumor tissue. However, the mice bearing the tumor excreted extremely large amounts of calcium in their urine, and their bony tissues contained less calcium and phosphorus than controls. Moreover, histology of bony tissues of these nude mice clearly demonstrated the decrease in trabecular tissues and cortical thickness as well as remarkable activation of osteoclasts. Another patient with a CSF-producing bronchogenic squamous cell carcinoma showed mild granulocytosis and hypercalcemia. The biopsied tumor tissue was transplanted into nude mice, which developed marked granulocytosis (300,000/microliters) and also severe hypercalcemia (18 mg/dl). These results suggest the presence of a new syndrome of granulocytosis and hypercalcemia associated with CSF-producing tumors. The causal mechanism of the hypercalcemia was shown to be some humoral factor which activates osteoclasts other than parathyroid hormone. Neither prostaglandins nor osteoclast-activating factor seemed to be the cause of the hypercalcemia.

Purification of a calcium-activated neutral proteinase from human placenta.

A calcium-activated neutral proteinase has been purified to homogeneity from human placenta. The purified enzyme is a dimer composed of Mr 73 000 and 30 000 subunits. Half-maximal activity is observed at 250 microM Ca2+. It requires reduced sulfhydryl groups and neutral pH for optimal activity. Leupeptin, antipain, E-64, sulfhydryl-blocking agents and endogenous proteinase inhibitor inhibit the purified enzyme. This paper is the first to describe the purification and characterization of a calcium-activated neutral proteinase from a human non-muscular parenchymatous organ.

Inhibition of migration and proliferation of vascular smooth muscle cells by dehydroepiandrosterone sulfate.

Dehydroepiandrosterone (DHEA) and its sulfate (DHEA-S) are the most abundant steroids in humans, and their serum concentrations progressively decrease with age. Although relationships between DHEA(-S) and many age-related illnesses have been postulated, the mechanisms for their effects remain unknown, and specific receptors for these molecules have not been identified. In this paper, to investigate the role of DHEA(-S) in atherogenesis, we studied the proliferation and migration of a rabbit vascular smooth muscle cell line, SM-3, in the presence of DHEA(-S). Cellular proliferation was inhibited by DHEA-S, and to a lesser extent by DHEA. Modified Boyden's chamber assays revealed that DHEA-S inhibited the migration of SM-3 cells toward PDGF-BB. In cell attachment assays, DHEA-S inhibited the attachment of SM3 cells to fibronectin. It was suggested that the inhibitory effect of DHEA-S for SM-3 proliferation and migration was due to the decreased interaction with fibronectin. Scatchard analysis revealed the presence of two populations of DHEA-S binding sites in the nuclear fraction, and a smaller number in the cytosolic fraction. Since the dissociation constant of the higher affinity site was similar to the serum DHEA-S concentration in humans (Kd = 5.8 microM), this binding site could be functional under physiologic conditions. These findings suggest that there may be receptor-mediated anti-atherogenic actions of DHEA-S.

Hepatic and renal expression of rat apolipoprotein E under control of the metallothionein promoter in transgenic mice.

We created three lines of transgenic mice with an integrated rat genomic apolipoprotein E gene fused with the mouse metallothionein I promoter. These lines transcribed rat apoE mRNA in the liver and/or in the kidney and expressed significant amounts of rat apoE in plasma. Enhancement of the plasma level by treatment with Zn ion or Bi ion was observed.

Coronary angioplasty of chronic total occlusions with bridging collateral vessels: immediate and follow-up outcome from a large single-center experience.

OBJECTIVES: The purpose of the present study was to assess the effect of bridging collateral vessels on the success of coronary angioplasty of chronic total occlusions in the context of state of the art technology and operator skill. BACKGROUND: Coronary angioplasty of chronic total occlusions has been associated with relatively low success rates. Because the presence of bridging collateral vessels in chronic total occlusion has been reported to be the major predictive factor in procedural failure, angioplasty is often not recommended in patients with such vessels. METHODS: Three hundred ninety-seven consecutive patients undergoing coronary angioplasty for chronic total occlusion were classified into two groups. Patients in group I had chronic total occlusion with bridging collateral vessels (97 patients, 109 total occlusions), and patients in group II had chronic total occlusion without such vessels (300 patients, 324 total occlusions). RESULTS: The mean +/- SD duration of occlusion was 46 +/- 66 months (range 2 to 170) in group I and 27 +/- 39 months (range 2 to 112) in group II (p < 0.05, high power value 0.83, group I vs. group II). Angioplasty for single-vessel disease was performed in a smaller proportion of patients in group I than in group II (22% vs. 36%, p < 0.05; power value 0.77). Procedural success was achieved in 82 chronic total occlusions in group I and 270 chronic total occlusions in group II (75% vs. 83%, p = 0.07; power value 0.53). The rates of restenosis and reocclusion were 54% and 16%, respectively, for group I and 56% and 13%, respectively, for group II (p = 0.76, 0.46; power value 0.51, 0.47). Complications were minor with no Q wave infarction or requirement for urgent bypass surgery in either group. Of 81 patients with unsuccessful coronary angioplasty, 1 patient from group I (1%) and 3 patients from group II (1%) required pericardiocentesis because of cardiac tamponade. Guide wire manipulation did not impair the flow of bridging collateral channels in group I. CONCLUSIONS: Coronary angioplasty can open chronic total occlusions, with or without bridging collateral channels, for safe and effective recanalization without major complications.

The effect of ACTH administration on serum estrogens, LH, and FSH in the aged.

The effect of administration of long acting ACTH on serum levels of estrogens, LH and FSH was studied in aged subjects. Nine women and 5 men between 66 and 90 years of age were examined. One milligram of long-acting synthetic ACTH was given in every 12 h for 2 days. Significant increases in serum estrone and estradiol levels were induced in parallel with serum cortisol at 24 hours and no further change was observed at 48 hours. The estrone/estradiol ratio increased from a control value of 3.0 to 4.0 at 24 hours and to 4.7 at 48 hours (P less than 0.02) compared to control values.

Radioreceptor assay for insulin.

A sensitive and simple radioreceptor assay (RRA) capable of detecting 10 muU/ml of serum insulin has been developed using the crude membrane fraction of guinea pig kidney and 125I-porcine insulin. Using this assay system, serum insulin during 100 g oral glucose tolerance tests in six males including one obese man was measured. The mean concentration of serum insulin increased from 19 muU/ml during fasting to maximum of 90 muU/ml after 60 min of glucose load and then gradually declined to 38 muU/ml by 180 min. The values obtained by the present RRA agreed well with immunoreactive insulin (IRI).

Familial unresponsiveness to thyrotropin by autosomal recessive inheritance.

Unresponsiveness to TSH has been identified and sufficiently studied in only three patients. We report siblings with this defect as the first documentation of familial occurrence. A 26-yr-old woman was diagnosed with congenital hypothyroidism during infancy. The thyroid was atrophic, and thyroid function tests without T4 replacement showed serum free T4 levels below 3 pmol/L, serum TSH of 125 mU/L, and serum thyroglobulin below 5 mg/L. 123I scintigram showed decreased uptake (5% at 24 h), but normal shape at the correct position in the neck. Autoantibodies against thyroglobulin, thyroid peroxidase, and TSH receptor in serum were not detected. The amount of cAMP released into FRTL-5 cell culture in the presence of TSH from the patient was not different from that released by the same amount of TSH from normal subjects, suggesting that TSH bioactivity in our patient was normal. The brother of the patient also had congenital hypothyroidism, and the data on his thyroid function was similar to that for his sister. There was a consanguineous marriage in the parents of the siblings, and the mother of the patients had a normal serum free T4 level, but slightly increased serum TSH and thyroglobulin levels, indicating subclinical hypothyroidism. The possible pathogenesis of TSH unresponsiveness in our patients includes a mutation in the TSH receptor gene, abnormality in transcription-regulating factor, abnormality in GTP-binding protein, and/or inhibition of the action of cAMP. The family history of the patients suggests that the mode of inheritance in TSH unresponsiveness is autosomal recessive.