Puromycin-induced necrosis of crypt cells of the small intestine of mouse.

Chemical and radioautographic analysis of the small intestine of mice injected intraperitoneally with puromycin revealed an immediate decrease of precursor incorporation into DNA and protein and a delayed decrease of precursor incorporation into RNA. In addition to this decrease of precursor incorporation, damage to the crypt cells, but not to the cells of the villus of the small intestine, was observed. Further examination of other dividing cells (spleen) and nondividing cells (liver and heart) of these mice showed again that only cells of actively dividing tissues were damaged. The metabolic inhibitors actinomycin D, cytosine arabinoside, actidione, and puromycin aminonucleoside were used in an attempt to clarify the mechanism of cell damage by puromycin. The results showed that there was no clear correlation between cell necrosis and the pattern of inhibition of synthesis of DNA, RNA, or protein.

Cytochalasin B. VI. Competitive inhibition of nucleoside transport by cultured Novikoff rat hepatoma cells.

Cytochalasin B competitively inhibits the transport of uridine and thymidine by Novikoff rat hepatoma cells growing in suspension culture with apparent K(i)'s of 2 and 6 microM, respectively, but has no effect on the intracellular phosphorylation of the nucleosides. Choline transport is not affected by cytochalasin B. Results from pulse-chase experiments indicate that cytochalasin B has no direct effect on the synthesis of RNA, DNA, or uridine diphosphate-sugars. The inhibition of uridine and thymidine incorporation into nucleic acids by cytochalasin B is solely the consequence of the inhibition of nucleoside transport.

Chemopreventive effects of myo-inositol and dexamethasone on benzo[a]pyrene and 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone-induced pulmonary carcinogenesis in female A/J mice.

The objective of the present investigation was to prevent cancer of the lung by use of chemopreventive agents. Administrations of diets containing added myo-inositol or dexamethasone singly or in combination (the latter being the most potent) are being studied for this purpose. In previous work, the two compounds were shown to inhibit benzo(a)pyrene [B(a)P]-induced pulmonary adenoma formation in female A/J mice when fed during the postinitiation period [ie., starting 1 week after the last of three administrations of B(a)P by oral intubation]. In the present investigation, a longer administration schedule was used, which encompasses both the initiation and the postinitiation stages of carcinogenesis. The feeding of the test compounds was started 2 weeks prior to the first dose of carcinogen and continued for the duration of the experiment. Under these conditions, reductions in tumor formation were: myo-inositol, 64%; dexamethasone, 56%; and both together, 86% (P < 0.001 for all three). Addition of both compounds resulted in the largest inhibition that has been achieved with this experimental model as used in these investigations. Studies have begun of inhibition of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced pulmonary adenoma formation by myo-inositol and dexamethasone. The two compounds inhibit pulmonary carcinogenesis when fed singly or in combination. When fed throughout the entire protocol, reductions in tumor formation were: myo-inositol, 46%; dexamethasone, 41%; and both together, 71% (P < 0.001 for all three). The results of these investigations demonstrate that myo-inositol and dexamethasone inhibit pulmonary adenoma formation resulting from exposures to two major pulmonary carcinogens, B(a)P and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone.

Analysis of human promyelocytic leukemia cell (HL60) variants insensitive to phorbol ester tumor promoters.

The cells of the human promyelocytic leukemia cell line (HL60) stop growing and differentiate into macrophage-like cells when exposed to nM concentrations of the phorbol ester tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). By exposing cells to the frameshift mutagen ICR-191 and subsequently selecting for resistance to the differentiating effects of nM amounts of TPA, we have isolated TPA-insensitive variants. These variants can grow in up to 320 nM TPA concentrations and do not differentiate into morphologically or functionally mature macrophages. The number of phorbol ester receptors, their affinity for phorbol dibutyrate, and the regulation of receptors are the same as for wild-type HL60 cells. As the resistance to TPA increases in the variants, so does the number of cells with increased ploidy. Wild-type HL60 cells are nearly 100% hypodiploid with a modal chromosome number of 43, while a partially TPA-resistant variant (DM30) has 30% hyperdiploid cells with a mean chromosomal number of 70, and a completely resistant variant (DM90) is 93% hyperdiploid averaging 74 chromosomes/cell. The variants differentiate into neutrophils in response to dimethyl sulfoxide but are defective in respiratory burst activity as assayed by the reduction of the dye nitroblue tetrazolium. These variants could be useful in determining the mode of action of TPA in the promotion of tumors.

Binding of [3H]12-O-tetradecanoylphorbol-13-acetate to intact human peripheral blood lymphocytes.

Our studies indicate that tritiated 12-O-tetradecanoylphorbol-13-acetate ([3H]TPA) produced by the reduction of the C-20 aldehyde with sodium [3H]borohydride is recognized by the same cellular site as is unlabeled 12-O-tetradecanoylphorbol-13-acetate (TPA). None of the concentrations of TPA used in these studies had an effect on the cell number and viability of human peripheral blood lymphocytes (HPBL) when incubated up to 1 hr at temperatures of 37 and 4 degrees as compared to untreated controls. [3H]TPA was not significantly metabolized by these cells after 1 hr at 37 degrees. Examination of the binding of [3H]TPA with simultaneous examination of uptake of tritiated thymidine ([3H]dThd) in parallel cultures demonstrated a close correlation between the apparent binding constant (0.94 X 10(8) M-1) and the activation constant for TPA stimulation of [3H]-dThd incorporation (0.95 X 10(-8) M). Binding of [3H]TPA was examined in two experimental conditions in which TPA-induced mitogenesis was inhibited: (a) preincubation of HPBL at 37 degrees for 24 hr causes a decrease of [3H]dThd uptake of 50% and an apparent loss of binding sites for [3H]TPA; and (b) glucocorticoid inhibition of [3H]dThd uptake in HPBL by 50%, however, did not reduce [3H]TPA binding. Our data suggest that cellular receptors either at the membrane or in the cytoplasm exist for TPA in HPBL. Alterations in binding of TPA to these receptors may account for the decrease in mitogenic response in preincubation experiments.

Characterization of integrated human papillomavirus type 11 DNA in primary and metastatic tumors from a renal transplant recipient.

A primary perianal squamous cell carcinoma and two metastatic tumors from a renal transplant recipient with a previous history of condyloma acuminatum were analyzed by filter hybridization for the presence of human papillomavirus (HPV) DNA. Each of the DNA extracts from these three tissues was found to contain HPV DNA. Stringent hybridization and restriction endonuclease analysis identified this viral DNA as HPV 11 related, which largely comigrated with cellular DNA, suggesting the presence of integrated viral DNA. Each DNA extract was analyzed by two-dimensional gel electrophoresis, which separates circular and linear forms of DNA and can demonstrate linear viral DNA, which comigrated with high molecular weight linear cellular DNA, thus implying viral integration. In all three cases the vast majority of viral DNA was found to comigrate with linear DNA; in addition, a significant portion comigrated with high molecular weight cellular DNA, suggesting the presence of integrated viral DNA in these tumors. Restriction endonuclease analysis of high molecular weight cellular DNA from each of these tumors revealed identical banding patterns, indicating that the integration site in each tissue is identical and, therefore, that all three tumors most likely originated from a single clonal event. These molecular results are presented in light of the clinical history of this patient with a histologically "low grade," but biologically aggressive, squamous cell carcinoma and suggest that HPV 11 may be associated with the initiation of malignant epithelial neoplasms.

Chemoprevention of pulmonary carcinogenesis by aerosolized budesonide in female A/J mice.

This investigation is part of a continuing effort to develop effective chemoprevention for carcinogenesis of the lung. The present study explores the use of aerosol administrations for this purpose. The agent selected for initial study was the synthetic glucocorticoid budesonide. This selection was based on previous work in which budesonide added to the diet was found to inhibit pulmonary adenoma formation in female A/J mice. However, high dose levels were required, i.e., of the order of 300 microg/kg, of body weight [L. W. Wattenberg and R. D. Estensen, Carcinogenesis (Lond.), 18: 2015-2017, 1997]. For aerosol administration of budesonide, a nose-only technique has been developed that entails nebulization of the compound dissolved in ethanol and subsequent stripping off of the solvent (less than 3 microl ethanol/liter of air remaining at the site of inhalation). The budesonide particles produced by the apparatus had a mass median aerodynamic diameter of less than 1 microm. An experiment has been carried out in which the inhibitory effects of aerosolized budesonide, given for 1 min six times a week, were studied. Concentrations of budesonide of 26, 81, and 148 microg/liter of air (calculated doses of 23, 72, and 126 microg/kg of body weight) were used. The aerosols were started 1 week after three oral administrations of benzo(a)pyrene (2 mg/20 g of body weight) to female A/J mice. All three doses of budesonide resulted in more than 80% inhibition of pulmonary tumor formation compared to the aerosol control and 90% or greater compared to mice not exposed to aerosol. The difference in inhibition is due to the aerosol procedure itself, which produces a reduction in tumor formation. A decrease in splenic weight (evidence of a systemic effect) occurred at all doses of budesonide. To the best of our knowledge, this is the first published effort at the use of aerosol administration to prevent neoplasia of the respiratory tract. The results of the present study show that administration of a potential chemopreventive agent by aerosol at a low dose can inhibit the occurrence of pulmonary carcinogenesis in female A/J mice.

Association of Ki-ras with amplified DNA sequences, detected in human ovarian carcinomas by a modified in-gel renaturation assay.

A modified in-gel DNA renaturation technique, which detects DNA sequences amplified greater than 7-fold in human DNA, was used to analyze gene amplification in surgical specimens of primary and metastatic ovarian carcinomas. Amplified DNA sequences were detected in two of eight tumors. Hybridization of these samples with different oncogene probes revealed that both tumors contained an amplified Ki-ras gene, which in one case was coamplified with c-myc. In one of the tumors, Ki-ras was found to be amplified in both the primary tumor and three different metastatic nodules. No mutations at codons 12 or 61 of Ki-ras were detected in these tumors. No additional cases of Ki-ras or c-myc amplification were detected by Southern hybridization in the tumors that were found to be amplification negative by modified in-gel renaturation assays. These results indicate that gene amplification in ovarian carcinomas is likely to involve the Ki-ras oncogene.

Concanavalin A and alloxan interactions on glucose-induced insulin secretion and biosynthesis from islets of Langerhans.

The pretreatment of isolated islets of Langerhans with concanavalin A (Con A) completely blocks alloxan from suppressing the insulin release response to glucose. The lectin itself inhibits insulin secretion. This effect is dose dependent and reversible. Con A, however, has no protective action against the inhibition of glucose-induced insulin biosynthesis in islets exposed to alloxan. The protective action of Con A on alloxan toxicity is likely to be at the beta-cell surface at a membrane recognition site for glucose as a stimulus for secretion. The insulin biosynthetic effect of glucose appears to be mediated through a separate mechanism.

Phorbol myristate acetate: effect of a tumor promoter on insulin release from isolated rat islets of Langerhans.

Phorbol myristate acetate (PMA), a tumor-promoter capable of influencing biologic functions of many cell systems, has been demonstrated to augment glucose-initiated insulin release from isolated rat islets of Langerhans. PMA caused a 2-fold increase in insulin release. This effect of PMA did not alter the sigmoidal relationship of insulin released to glucose concentration. The effect of PMA on insulin secretion from the islet beta-cells persists and a challenge with glucose alone, subsequent to a pulse of PMA, elicits an augmented insulin release response.

N-acetylcysteine suppression of the proliferative index in the colon of patients with previous adenomatous colonic polyps.

This investigation is part of an effort to develop chemoprevention for carcinogenesis of the large bowel. The agent investigated is N-acetylcysteine (NAC). We used as a predictive biomarker, the proliferative index (PI), in a short-term human study. Patients with previous adenomatous colonic polyps are a cohort with increased risk for colon cancer and an increased PI of colonic crypts. They were randomly assigned to an experimental group given 800 mg/day of NAC for 12 weeks or a placebo group. Using proliferative cell nuclear antigen immunostaining, the PI of colonic crypts was measured prior to and after the treatments. The PI of the NAC group was decreased significantly (P < 0.02) while the placebo group showed no difference (P > 0.45). Since this decrease in PI may be an indicator of decreased risk of colon cancer, more extensive studies of the potential of NAC as a chemopreventive agent for colon cancer appear warranted.

Intravascular lymphomatosis (malignant angioendotheliomatosis) presenting as pulmonary hypertension.

Intravascular lymphomatosis is a rare lymphoma characterized by proliferation of malignant cells within the lumen of small blood vessels. We describe a case of intravascular lymphomatosis resulting in pulmonary hypertension, hypoxemia, and dyspnea. This lymphoma occasionally responds to combination chemotherapy, suggesting that pulmonary hypertension secondary to intravascular lymphomatosis may be reversible. Intravascular lymphomatosis should be considered in the differential diagnosis of pulmonary hypertension.

Modulation of human neutrophil chemotactic responses by cyclic 3',5'-guanosine monophosphate and cyclic 3',5'-adenosine monophosphate.

Cyclic 3',5'-guanosine monophosphate (cGMP) and cyclic 3',5'-adenosine monophosphate (cAMP) and compounds known to effect the intracellular concentrations of these nucleotides were examined for their ability to effect human neutrophil (PMN) responsiveness to chemotactic stimulation. Incubation of neutrophils with agents recognized to promote increases in intracellular cAMP in a variety of tissues (i.e., epinephrine, norepinephrine, isoproterenol, histamine, cholera toxin, and prostaglandin E-1 and E-2) or with cAMP inhibited the leukotactic response to a bacterial chemotactic factor. In contrast, cGMP and compounds which have been shown to promote increases in intracellular cGMP concentration (i.e., acetylcholine, carbamylcholine, phorbol myristate acetate, and prostaglindin F-2-alpha) markedly enhanced the neutrophil chemotactic response. The inhibitory or stimulatory influences on chemotactic responsiveness promoted by several of the agents could be shown to be blocked by a specific pharmacologic antagonist of the particular compound tested. These data support the hypothesis that cGMP and cAMP can provide opposing regulatory influences on certain cellular functions; in this case, directed motility of leukocytes.

Phorbol myristate acetate: is a tumor promoter acting as a hormone?

Tumor promoters act on carcinogen-initiated tissues to cause phenotypic expression of malignancy. Phorbol ester tumor promoters, like hormones, act on cells and tissues at nanomolar concentrations, often producing "physiological" effects. These promoters also act on cells to produce what appear to be nonphysiologic or toxic effects. One of the major questions regarding these phenomena is, Which action or actions of promoters are important in phenotypic expression of malignancy?

Studies of chemopreventive effects of myo-inositol on benzo[a]pyrene-induced neoplasia of the lung and forestomach of female A/J mice.

There is a continuing effort at identifying chemopreventive agents that might be useful in preventing cancer of the lung. In the present study, the effects of myo-inositol and dexamethasone on benzo[a]pyrene (B[a]P)-induced pulmonary adenoma formation in female A/J mice was investigated. A diet containing 3% myo-inositol fed beginning 1 week after B[a]P administration reduced the number of pulmonary adenomas by 40% but did not prevent forestomach tumors, which also occur in this experimental model. Under the same conditions, dexamethasone, 0.5 micrograms/g diet, inhibited pulmonary adenoma formation by 57% and also inhibited forestomach tumor formation to a similar extent. Feeding a diet containing both myo-inositol and dexamethasone resulted in an additive effect on the inhibition of pulmonary adenoma formation. The combination of myo-inositol plus dexamethasone produced almost identical inhibition of forestomach tumor formation to that of dexamethasone alone. The results of the present study are preliminary, but may provide a basis for future investigation into strategies for chemoprevention of pulmonary neoplasia.

Lack of correlation between effects of tumor promoter TPA on plasminogen activator production, phosphatidyl choline synthesis, and hexose transport in mammalian cell culture systems.

We have investigated the effects of the tumor promoter 12-0-tetradecanoyl-phorbol-13-acetate (TPA) on plasminogen activator production, hexose transport and metabolism, and the incorporation of choline into the acid soluble pool and into phosphatidylcholine in suspension cultures of mouse L, mouse P388 leukemia, human HeLa, and Chinese hamster ovary cells, and in monolayer cultures of baby hamster kidney (BHK), mouse 3T3, mouse 3T6, and mouse P388D1 macrophage-like cells. BHK, 3T3, P388D1, and P388 cells produced plasminogen activator constitutively, but no significant production was observed in the other cell lines. Plasminogen activator production was induced or stimulated by TPA only in P388 cells (10- to 20-fold by 100 ng TPA/ml). On the other hand, phosphatidylcholine synthesis was stimulated by TPA only in HeLa cells, and hexose transport, as measured with 3-0-methyl-D-glucose, only in 3T3 and P388D1 cells, as well as in human lymphocytes. The stimulation of hexose transport occurred more rapidly than the induction of plasminogen activator production and seemed to be part of the mitogenic response of cells to TPA treatment. A stimulation of deoxyglucose uptake was similarly limited to 3T3 and P388D1 cells. A significant decarboxylation of carbon 1 of deoxyglucose occurred in P388 and P388D1 cells, but not in Novikoff cells, and any decarboxylation that occurred was not stimulated by TPA. The results indicate that the various investigated responses of cells to TPA are unrelated and occur independent of each other. The time courses of the biochemical responses also differ significantly.

Introduction of a heterologous nucleus into enucleated cytoplasms of cultured mouse L-cells.

Mouse L-cells exposed to cytochalasin B undergo random nuclear protrusion and, occasionally, total enucleation. The anucleate cell remains viable for several days. Chick erythrocytes were fused to L-cells with inactivated Sendai virus. The resulting hybrids, containing one or more erythrocyte nuclei in addition to the L-cell nucleus, were treated with cytochalasin B. Through the process of random enucleation, the L-cell nucleus was extruded from some hybrid cells while the erythrocyte nucleus was retained in the hybrid. In other experiments, chick erythrocytes were fused directly to enucleated L-cells. The result in both instances is the introduction of an erythrocyte nucleus into L-cell cytoplasm. The erythrocyte nucleus swells and the hybrid cell is capable of incorporating radioactive uridine.