Henryk Taper

Effects of sodium ascorbate (vitamin C) and 2-methyl-1,4-naphthoquinone (vitamin K3) treatment on human tumor cell growth in vitro. I. Synergism of combined vitamin C and K3 action

The effects of sodium ascorbate (vitamin C) and 2‐methyl‐1,4‐naphthoquinone (vitamin K3) administered separately or in combination on the in vitro cultured human neoplastic cell lines MCF‐7 (breast carcinoma), KB (oral epidermoid carcinoma), and AN3‐CA (endometrial adenocarcinoma) have been examined. When given separately, vitamin C or K3 had a growth inhibiting action only at high concentrations (5.103 μmol/1 and 105 nmol/1, respectively). Combined administration of both vitamins demonstrated a synergistic inhibition of cell growth at 10 to 50 times lower concentrations. At this level separately given vitamins are not toxic. The sensitivity to this treatment was somewhat different in the three cell lines, being slightly higher for KB line. This tumor cell growth inhibitory effect was completely suppressed by the addition of catalase to the culture medium containing vitamins C and K3, suggesting an excessive production of hydrogen peroxide as being implied in mechanisms responsible for the above‐mentioned effects.

The effect of estradiol-17-phenylpropionate and estradiol benzoate on N-nitrosomorpholine-induced liver carcinogenesis in ovariectomized female rats.

The influence of synthetic estrogens on the N‐nitrosomorpholine (NNM)‐induced liver carcinogenesis in ovariectomized young adult female rats was investigated and compared to rats which received only the carcinogen or estrogens. Estrogens when chronically administered after the cessation of carcinogen treatment increased the carcinogenic effect of NNM. In such conditioned animals the number of nodules per number of rats was 23/31, that of hepatocellular carcinomas 9/31, whereas in animals which received only the hepatocarcinogen the incidence of nodules and carcinomas in liver was respectively 11/31 and 3/31. Higher incidence of benign and malignant tumors in other organs was also observed in these animals. Rats which received a single dose of estrogens simultaneously with NNM developed slightly fewer tumors in liver and in other organs. Since under my experimental conditions the long‐term treatment with synthetic estrogens alone did not induce any focus, nodule or hepatocellular carcinoma in the liver, I suggest that the estrogens were acting rather as tumor promotors than true initiators of liver carcinogenesis.

In vivo reactivation of DNases in implanted human prostate tumors after administration of a vitamin C/K(3) combination.

Human prostate cancer cells (DU145) implanted into nude mice are deficient in DNase activity. After administration of a vitamin C/vitamin K3 combination, both alkaline DNase (DNase I) and acid DNase (DNase II) activities were detected in cryosections with a histochemical lead nitrate technique. Alkaline DNase activity appeared 1 hr after vitamin administration, decreased slightly until 2 hr, and disappeared by 8 hr after treatment. Acid DNase activity appeared 2 hr after vitamin administration, reached its highest levels between 4 and 8 hr, and maintained its activity 24 hr after treatment. Methyl green staining indicated that DNase expression was accompanied by a decrease in DNA content of the tumor cells. Microscopic examination of 1-μm sections of the tumors indicated that DNase reactivation and the subsequent degradation of DNA induced multiple forms of tumor cell death, including apoptosis and necrosis. The primary form of vitamin-induced tumor cell death was autoschizis, which is characterized by membrane damage and the progressive loss of cytoplasm through a series of self-excisions. These self-excisions typically continue until the perikaryon consists of an apparently intact nucleus surrounded by a thin rim of cytoplasm that contains damaged organelles.

Reversibility of acid and alkaline deoxyribonuclease deficiency in malignant tumor cells.

The nature of DNAse deficiency, which appears to be characteristic for malignant tumor cells, was investigated by the histochemical lead nitrate technique under various experimental conditions. Reappearance of distinct alkaline and acid DNAse activity was observed on the periphery of spontaneously occurring tumor necrosis, at early stages of the in vitro induced tumor necrosis, in necrotic tumor cells after in vivo irradiation and after in vitro treatment with different compounds. A membrane releaser did not reactivate DNAses in viable tumor cells, whereas the homogenate from tumor tissue inhibited DNAses in normal rat liver. These findings indicate that alkaline and acid DNAse deficiency in malignant tumor cells is a reversible phenomenon. This reversal of enzymatic activity has different histochemical and chronological patterns and specific reactivating factors for each DNAse. The masking effect of DNAse activity in malignant tumor cells is probably linked to natural enzyme inhibitors and its reversal to early stages of tumor necrosis.

Activity of alkaline and acid nucleases in tumors of the human central nervous system: Histochemical study

The alkaline and acid DNAse and RNAse were histochemically investigated in the tumors of the human central nervous system. In most of the malignant tumors, none of the four types of nucleases manifested any activity, whereas in the benign tumors this activity was similar to that of those normal tissues from which they originated. The necrotic areas of malignant tumors had a peripheral zone in which the activity of the nucleases reappeared. This reappearance was probably due to the splitting of the nuclease‐inhibitor complex. The significance of nucleases in malignancy and their possible role in the protection of the genetic stability of the cell are briefly discussed.

Increase in nuclease activity as a possible means for detecting tumor cell sensitivity to anticancer agents

Histochemical activity of nucleases was investigated in sensitive and resistant rat tumors at several periods after a single dose of cyclophosphamide and was compared with that in a control group of untreated tumors. Intense increase of nuclease activity (mainly that of acid DNAse) appeared only in the cells of the sensitive tumors at 12 hours, reaching its maximum three days after drug administration. Untreated malignant tumors usually have variable amounts of spontaneous necrosis with high nuclease activity. Not the absolute values of high nuclease activity but only the increase of such activity from the basal level of untreated tumors to that measured at frequent periods after trial treatment may be suggested as an early marker of therapeutically induced tumor cell necrosis. However, more investigation is needed before such a marker might be applied as a test for tumor sensitivity to therapeutic agents.

Histochemical correlation between glycogen, nucleic acids and nucleases in pre-neoplastic and neoplastic lesions of rat liver after short-term administration of N-nitrosomorpholine

After 7 weeks of oral administration of the carcinogen N-nitrosomorpholine (12 mg NNM ad 100 ml of drinking water) to male rats, marked hepatocellular changes were found predominantly in the centers of the lobules. These were loss of glycogen, disorganisation of the basophilic bodies and, sometimes, loss of cytoplasmic basophilia or pyroninophilia, necrotic cells and increase in the activity of acid DNA se and RNA se. These centrilobular alterations were reversible after withdrawal of the carcinogen. They are, therefore, attributed to the nonspecific-toxic effect of the carcinogen. In peripheral and midzonal regions of the lobules basically different cellular changes appeared which were unimportant during the phase of intoxication, but became prominent after cessation of the carcinogenic treatment. These lesions were: excessive storage of glycogen, displacement of basophilic bodies and an increase in cytoplasmic acidophilia. The hepatocytes showing these cytoplasmi changes initially formed foci. Neoplastic nodules and frank hepatocellular carcinomas developed later. During these later stages of the experiment both the foci and the nodules consisted of 4 main types of altered hepatocytes: 1) “clear” glycogen storage cells, 2) acidophilic cells, 3) vacuolated (fat storing) cells, 4) basophilic (pyroninophilic) cells poor in, or free from, glycogen. The larger nodules and carcinomas contained predominantly basophilic cells. The activity of nucleases, especially that of acid DNA se, decreased in small foci which as a rule developed later than the foci of glycogen storage. In most cells of neoplastic nodules and carcinomas the activity of these enzymes disappeared, but it reappeared in necrotic cells. The progressive alterations in the activity of the nucleases seemed to be related to the phenotypic expression of malignancy and could be a sign of a fundamental metabolic change taking place during a relatively late step in the malignant transformation. Die orale Gabe des Carcinogens N-Nitrosomorpholin (12 mg ad 100 ml Trinkwasser) an männliche Ratten führt innerhalb von 7 Wochen zu ausgeprägten, vorwiegend acinuszentralen Veränderungen der Hepatocyten: Glykogenschwund, Disorganisation der Ergastoplasmaschollen, in manchen Zellen Verlust der cytoplasmatischen Basophilie oder Pyroninophilie, Nekrosen, Zunahme der Aktivität der sauren DNAse und RNAse. Nach Absetzen des Carcinogens erweisen sich diese Zellveränderungen als reversibel. Sie werden daher auf die unspezifisch-toxische Wirkung des Carcinogens zurückgeführt. In peripheren und intermediären Läppchenregionen kommt es zu grundsätzlich anderen Zellveränderungen. Sie sind während der Vergiftungsphase unbedeutend, treten nach Absetzen des Carcinogens aber bei allen Tieren deutlich hervor: übermäßige Glykogenspeicherung, Dislokation der Ergastoplasmaschollen, Zunahme der cytoplasmatischen Acidophilie. Hepatocyten mit diesen Cytoplasmaalterationen bilden zunächst Herde. Später entwickeln sich neoplastische Knoten und ausgeprägte hepatocelluläre Carcinome. In den späten Versuchsstadien bestehen sowohl die Herde als auch die neoplastischen Knoten im wesentlichen aus 4 verschiedenen cellulären Schädigungstypen: 1. „Klare” Glykogenspeicherzellen, 2. acidophile Zellen, 3. vacuolisierte (fettspeichernde) Zellen, 4. glykogenarme oder -freie basophile (pyroninophile) Zellen. Größere Knoten und Carcinome enthalten vorwiegend basophile Zellen. Die Aktivität der Nucleasen, speziell der sauren DNAse, geht in anfänglich nur kleinen peripheren und intermediären Läppchenbezirken zurück. Die Herde mit veränderter Enzymaktivität bilden sich in der Regel später aus als die Glykogenspeicherherde. In neoplastischen Knoten und Carcinomen zeigen die meisten Zellen einen vollständigen Verlust der Enzymaktivität, in nekrotischen Tumorzellen tritt die Enzymaktivität aber wieder in Erscheinung. Die fortschreitenden Veränderungen der Aktivität der Nucleasen scheinen eng mit der phänotypischen Ausbildung der Malignität verknüpft zu sein. Sie könnten eine grundlegende Umstellung des Zellstoffwechsels anzeigen, die sich in einem relativ späten Stadium der neoplastischen Zelltransformation ereignet.

Histochemical differences between so-called megalocytosis and neoplastic or preneoplastic liver lesions induced by N-nitrosomorpholine

Abstract Differences in histochemical pattern were found between the so-called megalocytes and preneoplastic and neoplastic hepatocytes after short-term administration of sublethal dose of N -nitrosomorpholine in rats. Megalocytes were poor in glycogen and intensely pyroninophilic. The activity of alkaline and acid nucleases was normal or increased. On the other hand the clear and acidophilic hepatocytes considered to be preneoplastic showed excessive storage of glycogen and had usually decreased or absent activity of nucleases. Basophilic cells of neoplastic nodules and hepatocellular carcinomas were practically free from glycogen and nuclease activity. The majority of megalocytes which disturbed seriously the lobular architecture of liver during the NNM intoxication, disappeared soon after the withdrawal of carcinogen. The megalocytes are interpreted as the result of an interaction between toxic and regenerative events.

Gastric carcinogenesis in rat induced by methylnitrosourea (MNU). morphology, and histochemistry of nucleases

27 squamous cell carcinomas of the forestomach, 2 adenocarcinomas of the glandular stomach and very numerous preneoplastic lesions of the mucosa in both parts of the stomach were induced after chronic oral administration of MNU in Wistar R rats. Single transplacental administration of MNU did not produce any gastric tumor, however preneoplastic alterations were detected in the forestomach of 42 animals and in the glandular stomach in 25 animals out of 51 examined, showing histologic patterns like those found in rats after chronic oral MNU administration. The pre- and neoplastic alterations in the forestomach demonstrated a distinct decrease of acid and alkaline DNase and RNase activity as compared with the normal forestomach. The last mentioned observation and our previously published results suggest that the deficiency of nucleases precedes or facilitates carcinogenesis. 27 Plattenepithelcarcinome des Vormagens, 2 Adenocarcinome des Drüsenmagens und sehr zalhreiche präneoplastische Veränderungen in der Schleimhaut beider Magenabschnitte wurden durch chronische orale Gaben von MNU bei Wistarratten hervorgerufen. Eine einzige transplacentare Gabe von MNU rief keine Magentumoren hervor, doch wurden unter 51 untersuchten Ratten in 42 Fällen im Vormagen und in 25 Fällen im Drüsenmagen praeneoplastische Veränderungen entdeckt, welche eine ähnliche histologische Beschaffenheit zeigten, wie die nach chronischer oraler Zufuhr von MNU. Die praeneoplastischen und neoplastischen Veränderungen im Vormagen zeigten eine deutliche Abnahme der sauren und alkalischen DNase- und RNase-Aktivität gegenüber der Norm. Diese Beobachtung und frühere Untersuchungsergebnisse lassen vermuten, daß ein Nucleasemangel die Carcinogenese erleichtert oder ihr vorausgeht.

Nucleases activity in different segments of the human digestive tube compared to the incidence of carcinomas (histochemical study)

SummaryThe alkaline and acid DNase and RNase activity was histochemically investigated in biopsies from the human digestive tube. Activity of these enzymes in the mucosal epithelium in different segments of the digestive tube was compared to the statistical incidence of malignant tumors deriving from this tissue (carcinomas). It was found that the alkaline and acid nucleases activity was very intense in small intestine precisely in this segment where the incidence of carcinomas was low, whereas the low activity of these enzymes in the stomach and large intestine corresponded to the high incidence of carcinomas. This observation confirmed our previously elaborated hypothesis, according to which the low activity of nucleases in normal tissues appeared to be a predisposing factor for malignant transformation.It could be also supposed that the nucleases constitute some kind of double barrier mechanism protecting the genetical stability of the cell against foreign nucleic acid incorporation or production; alkaline nucleases being an extracellular and acid nucleases an intracellular barrier.