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[Tumor prevention by vitamin A: in vitro studies with human cultured fibroblasts (author's transl)].

Following the well established prevention of chemical carcinogenesis by vitamin A in several species of laboratory animals, we performed in vitro studies with human diploid fibroblasts in culture. Vitamin A-palmitate, alltransretinoic acid and the analogue compound Ro 10-9359 were found to reduce the formation of active intermediates following the administration of G3H Benzo(a)pyrene to the cells. This effect which lead to a considerable decrease of alkylated DNA is not based on a direct inhibition of Benzpyrene metabolizing enzymes by the retinoids but by a preferential inhibition of the de novo synthesis of these enzymes. This caused the well known substrate mediated enzyme induction of benzpyrene metabolizing enzymes to cease. From our data we conclude that the mechanism of the cancer protective effect of vitamin A with respect to certain carcinogens is based on an inhibited activation of procarcinogens. This effect can also be expected in human tissues.

Benzopyrenes

In vitro studies of human breast carcinoma cells obtained by aspiration biopsy.

Cells from 14 human breast carcinoma were obtained by aspiration biopsy and used for in vitro cultivation. Growth could be observed in three cultures, which were derived from solid, partly adenoid growing tumors with metastases in the axillary nodes. The growth started after 2--6 days in culture and lasted 2--4 weeks. No permanent line could be established.

Adenocarcinoma

Amylin inhibits gastric cancer progression by targeting CCN1 and affecting the PI3K/AKT signalling pathway.

METHODS: This study used a combination of in vitro and in vivo experiments to investigate the role of amylin in the progression of GC. The expression of amylin in GC and its clinical correlation were evaluated using 38 pairs of GC and healthy human clinical samples. In vitro studies, human GC cell lines were treated with amylin to evaluate the effects of amylin on the proliferation, apoptosis and migration of GC cells. In in vivo studies, xenograft mouse models were established by subcutaneous injection of GC cells into nude mice, followed by treatment with amylin to assess tumor growth. Finally, Next-Generation Sequencing Technology (RNA-seq) was used to explore the potential mechanism of amylin on GC. RESULTS: We found that amylin expression was reduced in GC compared to adjacent normal gastric tissues and that elevated amylin expression was negatively correlated with adverse pathological factors (p&#x2009;<&#x2009;0.05). Additionally, we demonstrated that amylin impeded the growth, invasion, migration, and colony formation of GC cells and suppressed the epithelial-to-mesenchymal transformation of these cells (p&#x2009;<&#x2009;0.05). Tumour xenograft model experiments confirmed the tumour-suppressive effect of amylin in subcutaneous tumours in nude mice (p&#x2009;<&#x2009;0.05). Transcriptome sequencing (RNA-seq) revealed that amylin significantly down-regulated CCN1 gene expression in GC cells (p&#x2009;<&#x2009;0.001). Further intervention targeting CCN1 verified its significance as a target of amylin's anti-carcinogenic function in GC. Additionally, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed that amylin exerted its oncogenic effects by inhibiting the PI3K/Akt signalling pathway (p&#x2009;<&#x2009;0.05). CONCLUSIONS: Our findings demonstrate that amylin plays a crucial role in suppressing gastric cancer progression by targeting CCN1 and inhibiting the PI3K/Akt signalling pathway. These results suggest that amylin could serve as a potential therapeutic agent for GC treatment.

Humans

Chromosomal studies of human prostatic tumors in vitro.

Chromosomal studies were performed on primary cultures from human prostatic adenomas and carcinomas. All cells from both tumor types showed diploid chromosomal counts with a normal human karyotype. Chromosomal analysis was also performed on cells of the permanent epithelial line EB 33 derived from a human prostatic carcinoma in this laboratory. In contrast to the primary cultures, hypotriploid chromosome numbers were found during continuous culture for more than 1 year. The range of chromosome numbers increased with time in culture and was reduced to the original model number of 64 by only one animal passage ("nude" mice). Feulgen fluorometry on different passages of the permanent cell line EB 33 showed tetraploidy. This discrepancy to the hypotriploid chromosome number can be explained by different Feulgen hydrolytic properties of the different cell types.

Animals

In vitro studies of poison oak immunity. II. Effect of urushiol analogues on the human in vitro response.

Studies were performed to ascertain the effect of urushiol analogues on the in vitro lymphocyte blastogenesis elicited by urushiol in peripheral blood lymphocytes taken from individuals sensitized to poison oak or ivy. Urushiol is a mixture of alkylcatechols composed of a catechol ring coupled to mono-, di-, or tri-unsaturated C-15 or C-17 carbon side chains. Each of these two moieties, catechol ring and side chain, was tested for its role in eliciting reactivity. Analogues tested represented the catechol ring (3-methylcatechol), the mono- or di-unsaturated side chain (oleic or linoleic acid), and the saturated side chain coupled to a catechol ring (pentadecylcatechol), a blocked catechol ring (heptadecylveratrole), or a resorcinol (pentadecylresorcinol). Urushiol with a blocked catechol ring (urushiol dimethyl ether) was also included. Of these, only pentadecylcatechol evoked reactivity in sensitized lymphocytes, and this reactivity was only a fraction of that evoked by urushiol. This suggested that the system has some requirement for the side chain, and that the catechol ring is critical for reactivity. This was further investigated by testing the ability of some of these analogues to inhibit urushiol-specific blastogenesis. No inhibition was noted with compounds bearing the saturated side chain with modified ring structures (pentadecylresorcinol and heptadecylveratrole). However, both 3-methylcatechol and pentadecylcatechol (at equimolar concentrations) blocked reactivity. The results of our experiments suggested that although both the side chain and the catechol ring are required for reactivity, the latter is most critical. Unsaturation in the side chain is important for maximal reactivity because the saturated catechols were only partially as active as the urushiol oil. There may be a greater dose requirement for the catechol ring than for the side chain.

Alkenes

The response to stretch of human intercostal muscle spindles studied in vitro.

1. The discharge properties of human muscle spindles have been studied in vitro in a preparation based on the biopsied external intercostal muscle. 2. The static and dynamic responsiveness of thirty-six endings in twenty visualized and histologically identified spindles have been investigated using amplitudes and velocities of stretch likely to encompass those occurring in vivo. 3. The dynamic index, measured at a stretch velocity of 3 mm/sec, ranged from 3 to 40 impulses/sec and was distributed bimodally, consitent with the presence of primary and secondary endings. 4. The relationship between the dynamic index and the velocity of stretch was approximately linear both for primary and secondary endings up to the maximum velocity tested (10 mm/sec). 5. The frequency/extension relationship was approximately linear for both primary and secondary endings. The mean values of the slope for primary and secondary endings were 16-1 +/- 8-3 S.D. of the observation and 12-1 +/- 6-5 impulses/sec per five per cent extension. 6. The slopes of the frequency/extension relationship for endings lying in the same spindle were positively correlated, significant at the 10% level. 7. It was estimated from the results in vitro that the position sensitivity of human intercostal spindles in vivo ranges from 2 to 21 impulses/sec per millimetre.

Action Potentials

In-vitro studies of normal human thyroid cells: responses to thyrotrophin and dibutyryl cyclic AMP.

Follicular cells isolated from normal human thyroid tissue have been cultured for up to 140 h with bovine thyrotrophin (TSH) or dibutyryl cyclic AMP (DBcAMP). Both compounds induced marked reorganization of the cells into three-dimensional follicular structures, whilst non-supplemented cells assumed a monolayer form. Cultures treated initially with TSH or DBcAMP showed a greater iodide uptake capacity, in comparison with unsupplemented cultures, in which iodide uptake was markedly diminished after 24 h. The release of tri-iodothyronine (T3) and thyroxine (T4) into the medium was determined by radioimmunoassay. Both TSH- and DBcAMP-treated cells showed a significant increase in iodothyronine output compared with unsupplemented control cells. In contrast to the "classical" TSH-induced depression of the T4:T3 ratio in vivo, an increase in the ratio was observed for both TSH- and DBcAMP-supplemented cells in vitro. The ratio was also significantly greater after TSH than after DBcAMP, and possible implications of this findings are discussed.

Bucladesine

Autonomic receptor functions of the human ureter: an in vitro study.

The autonomic receptor functions of ureteral smooth muscle were studied in vitro on strips of human ureter obtained at surgery. A dose-dependent, reproducible, contraction response to 5-hydroxytryptamine (Serotonin, 5-HT) was demonstrated. This response could be blocked by methysergide. Responses to acetylcholine and to drugs stimulating adrenergic receptor functions were also found, but they were weak and inconsistent. This suggests that contraction of ureteral smooth muscle is mediated through receptors which are sensitive to 5-hydroxytryptamine.

Acetylcholine

Erythrocyte pH in respiratory and metabolic acid-base disturbances. Studies on human blood in vitro.

The influence of oxygenation in respiratory and metabolic acid-base disturbances on erythocyte pH has been studied on normal human blood "in vitro". In physiological pH range, pHe-pHi relationship is the same for PCO2 variations at constant metabolic level and for metablic level variations at constant PCO2. A "global" pHe-pHi relationship has been calculated at 0 and 100% SO2. The oxygen-linked pH shift depends on pH level and PCO2 and is different in plasma and erythrocyte. From these results arise conclusions on pHi and pHe dependency, physiological role of saturation level of hemoglobin and conditions of P50 normalisation at pH 7.4.

Acid-Base Imbalance

In vitro perfusion studies of the human placenta. VI. Evidence against active glucose transport.

Previous studies in our laboratory using "in vitro" perfusion have established that glucose transport across the human placenta is a carrier-mediated process. It is not known whether these carriers require the expenditure of metabolic energy to function. In the experiments presented here we demonstrate in the perfused placenta that there is not reduction in the rate of glucose transport or its analogue 3-O-methyl-alpha-D-glucopyranoside (3MG) in the presence of 10(-4) M dinitrophenol (DNP), an uncoupler of oxidative phosphorylation. The presence of DNP, however, does cause an increase in the glucose utilization rate as well as increased lactic acid production. In order to test whether glucose transport depends on the functioning of a sodium pump system, the sodium in the perfusion system was replaced with choline chloride. The final sodium content was 30 mEq/L. In the presence of a low sodium concentration there was no decrease in the rate of 3MG transport compared to the control experiments run at normal sodium levels. Also "counter transport" of glucose was observed, a further indication that the glucose carrier mechanism does not require a sodium gradient in order to function. Since the transport rate of glucose or its analogue 3MG across the placenta is not reduced in the presence of 10(-4)M DNP and is not reduced in the absence of a sodium gradient, it is unlikely that the mechanism of glucose transport is an active process requiring the expenditure of metabolic energy.

Adult

Trochanter bone marrow: a source of normal human colony forming cells.

In vitro studies of human hemopoiesis are often limited by the availability of normal bone marrow. We have overcome this difficulty by taking advantage of the bone marrow fragments removed during total hip replacement. We report here a comparative study of the colony forming capacity of trochanter and sternal or iliac crest marrow from five hematologically normal donors. Our data indicate that trochanter marrow is a reliable source of normal in vitro granulocyte/macrophage colony forming cells.

Bone Marrow Cells

NADPH production in the oxidative pentose phosphate pathway as source of reducing equivalents in glycolysis of human red cells in vitro.

Studies have been carried out on human erythrocytes in vitro to clarify the deficit of pyruvate formation under conditions when 2,3 DPG is degraded. The results lead to the conclusion that there exist a cross connection between the glycolytic and the oxidative pentose phosphate pathway which is mediated by the NADP/NADPH couple. NADPH serves as additional reducing equivalent in the reaction of the LDH. In the absence of glucose the pool of the metabolites of the pentose phosphate pathway is able to supply glucose-6-phosphate for the production of NADPH by recombination. The reaction of NADPH at the LDH is probably of significance under in vivo conditions.

Diphosphoglyceric Acids

Human insulinoma tissue: in vitro studies of proinsulin/insulin biosynthesis and release.

The rate of proinsulin/insulin turnover has been studied in human insulinoma tissue from ten patients. During the incubation of tumor tissue the ratio of immunoreactive insulin (IRI) release to content was significantly higher than that from isolated human pancreatic islets. The tumor cell cytoplasm (100,000 g supernatant S-100) contained approximately 45% of the IRI. Endogenous proinsulin and insulin were released throughout the incubation. Newly synthesized proinsulin was detected in the 15 min pulse microsomal, secretory granule, S-100 and media samples. These results suggest that defective hormonal storage and release mechanisms are operative in human insulinoma cells resulting in a higher turnover of proinsulin and insulin compared with pancreatic islet tissue.

Adenoma, Islet Cell

The permeability of the human ciliary and iridial epithelium to horseradish peroxidase. An in vitro study.

The permeability of the human ciliary epithelium to horseradish peroxidase (PO) has been studied in vitro with the electron microscope. Ciliary body and iris specimens were obtained from freshly enucleated eyes. PO was applied at the stromal side of the epithelium, and was left for 120 min. The movement of PO through the intercellular spaces of the human ciliary epithelium was blocked apically in the lateral intercellular spaces of the non-pigmented epithelial cells, indicating that these cells are girdled by zonulae occludentes. In the iridial epithelium, the same distribution pattern of peroxidase reaction products (PORP) was found, i.e. the progression of PO was blocked apically in the lateral intercellular spaces of the posterior epithelial cells. The study indicates that the human ciliary and iridial epithelium contains a system of zonulae occludentes, which represents a diffusion barrier to high molecular, water soluble substances. This is consistent with previous studies in several species of animals.

Adult