Effect of chemopreventive agents on separate stages of progression of benzo[alpha]pyrene induced lung tumors in A/J mice.

The effects of aerosol budesonide and dietary myo-inositol on progression of benzo[alpha]pyrene (B[alpha]P) induced carcinogenesis were studied in A/J mouse lung. First, we determined when to intervene in the carcinogenesis process by exposing several animals to B[alpha]P at 100 and 150 mg/kg of body wt. Groups of these animals were necropsied from 1 to 36 weeks post-carcinogen. The presence of different categories of lung tumors was noted over the 36 week time period. Hyperplasia first appeared approximately 6 weeks post-carcinogen followed by adenoma at 9 weeks, then by carcinoma at 26 weeks. From this temporal sequence we determined we could test for effects of preventive agents on progression to hyperplasia by intervening at 3 weeks, for effects on progression to adenoma by intervening at 6 weeks and for effects on progression to carcinoma by intervention at 12 weeks. Intervention at 3 weeks post-carcinogen with aerosolized budesonide delayed both hyperplasia and adenoma formation. Once hyperplasia appeared in budesonide treated animals, however, it increased at the same rate as in control animals, indicating a delay in progression. Progression from adenoma to carcinoma was reduced when budesonide was given 12 weeks post-carcinogen. Dietary myo-inositol failed to suppress progression from adenoma to carcinoma when started 12 weeks post-carcinogen. In summary, budesonide is a chemopreventive agent that has inhibitory effects on B[alpha]P induced carcinogenesis of the lung in A/J mice at all stages of progression from hyperplasia formation to cancer.

Chemoprevention of pulmonary carcinogenesis by brief exposures to aerosolized budesonide or beclomethasone dipropionate and by the combination of aerosolized budesonide and dietary myo-inositol.

This investigation is part of an effort to develop chemoprevention for carcinogenesis of the lung. It focuses on the efficacy of low doses of synthetic glucocorticoids administered either as single agents or in combination with a second compound, myo-inositol. Glucocorticoids are potent inhibitors of carcinogenesis. The use of low doses is important to avoid potential side-effects. The synthetic glucocorticoid budesonide, administered by aerosol for 20 s three times a week, was studied to determine its effects on benzo[a]pyrene-induced pulmonary adenoma formation in female A/J mice. Two dose levels were employed, 10 and 25 microg/kg body wt. The lower dose produced a 34% reduction in lung tumor formation and the higher dose level a 60% reduction in lung tumors. In additional groups of mice, the effects of 0.3% myo-inositol added to the diet was found to reduce pulmonary tumor formation by 53%. The two agents given in combination resulted in a greater inhibition of lung tumor formation than either by itself. Budesonide at 10 microg/kg body wt plus 0.3% myo-inositol reduced the number of tumors by 60% and budesonide at 25 microg/kg body wt plus 0.3% myo-inositol reduced lung tumor formation by 79%. To determine whether a glucocorticoid other than budesonide would have inhibitory effects in this experimental model, beclomethasone dipropionate administered by aerosol for 20 s three times a week was studied as a single agent and showed almost identical inhibitory properties to budesonide. The doses of the glucocorticoids calculated on a daily basis are within the range of those used widely for control of chronic allergic respiratory diseases in the human. The capacity of low doses of inhaled glucocorticoids to prevent pulmonary neoplasia and the enhancement of this preventive effect by myo-inositol, an essentially non-toxic compound, are findings that should encourage further work to evaluate the applicability of these agents to the prevention of neoplasia of the lung in the human.

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.

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

The objective of the present investigation was to determine conditions under which the synthetic glucocorticoid, budenoside, will inhibit benzo[a]pyrene (BaP)-induced pulmonary carcinogenesis when administered in the post-initiation period. For this purpose, female A/J mice were employed. The animals were given three administrations of 2 mg of BaP by oral intubation during a 1-week period. Budenoside was fed in the diet subsequent to the last dose of BaP. Using this format, two experiments were carried out to determine the effects of varying the time of administration of budenoside on the magnitude of the inhibition obtained. In both experiments, one group of mice was fed budenoside (1.5 mg/kg of diet) from 1 week after the last dose of BaP until the termination of the experiment, 15 weeks later. The reduction of pulmonary tumor formation under these conditions was 89% in the first experiment and 78% in the second (average 84%). In the first experiment the effects of feeding budenoside only during weeks 1-5 after BaP administration was studied. Under these conditions, inhibition of pulmonary tumor formation was 35%. In the second experiment, the effects of postponing the start of feeding budenoside was determined. In mice in which the budenoside feeding was delayed until 5 weeks after the last dose of BaP and then continued for the duration of the protocol, a 67% inhibition of tumor formation was found. The data obtained indicate that budenoside will produce inhibition of pulmonary adenoma formation when fed either early or late in the post-initiation stage of carcinogenesis, and that feeding throughout the entire post-initiation period gives maximum inhibition.

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.

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.

Effects of castration on plasminogen activator activities and plasminogen activator inhibitor type 1 in the rat ventral prostate.

The involution of the prostate gland after castration is an active process which requires the induction of new proteins. The plasminogen activator urokinase has been proposed to be a gene repressed by androgen which is activated upon castration and thus participating in the atrophy of the gland. However, urokinase is secreted by the ventral lobe of the rat prostate and this should be positively affected by androgens. The purpose of this study was to examine further the effects of castration upon plasminogen activator (PA) activities in the rat prostate and to determine possible explanations to this apparent dilemma. Castration of young sexually mature adult rats resulted in a substantial increase in PA activities at 4 days after castration in the ventral prostate, but then the activities returned to within the range of untreated animals with a longer duration of castration. Urokinase was the predominant molecular form of PA in the normal ventral prostate and it was the molecular form increased after castration; based upon its sensitivity to amiloride and its molecular size determined in zymograms. In contrast to the effect of castration, there was no increase in PA activities in the ventral prostate with treatment of rats with the antiandrogen flutamide, but rather a decrease when specific activity was expressed per unit DNA. In addition, the effect of castration was specific for the ventral lobe for there was no change in the PA activity in the dorsolateral prostate after androgen ablation. The diminished PA activities in the ventral prostates of rats castrated for 7 days or longer appeared to be due at least in part to an increase in plasminogen activator inhibitor type-1 (PAI-1). Immunoreactive PAI-1 was found predominantly in high molecular weight forms which indicates that the inhibitor was complexed with PA. Daily treatment of rats upon castration with agents known to retard the rate of regression of the involuting prostate gave dichotomous results. Hydrocortisone prevented the increase in PA activity, whereas treatment with actinomycin D, an inhibitor of RNA synthesis, not only did not prevent an increase in PA activity, but actually produced a superinduction in PA activity at 4 days orchiectomy. These data may be interpreted to mean that hydrocortisone stimulated PAI activity and that actinomycin D treatment blocked its induction. However, the actinomycin D data may also indicate that an increase in urokinase protein and mRNA after castration may result from some mechanism to conserve these molecules suggesting that this inhibitor of RNA synthesis prevented the transcription of messages for proteins involved in the degradation of urokinase message.

Effects of aging and castration on plasminogen activator and metalloprotease activities in the rat prostate complex.

Pathology of the prostate gland in rats and humans is associated with aging. Our objective was to examine the effects of aging on the activities of plasminogen activators and metalloproteases in the prostatic complex of rats. Plasminogen activator activities (very low in the anterior, lateral, and dorsal lobes, in contrast to higher activities in the ventral lobe of 4-month-old adult rats) increased with aging in the dorsal and anterior prostate lobes of 31-month-old rats; these activities also increased in the dorsal and lateral lobes upon castration of 18-month-old rats. The plasminogen activator activities in the ventral lobe did not increase with aging to 18 months but did increase 3-5-fold after castration of either young or old rats. Metalloprotease activities of 70 and 76 kDa were observed in the anterior and lateral lobes of 4 month untreated adult rats, whereas the dorsal lobe showed MP of 70 and 92 kDa. Castration of young adult rats increased activities of all three molecular forms of metalloprotease in these three lobes. Increased expression of metalloprotease activities was also found with aging to 31 months in the anterior, lateral, and dorsal lobes. However, changes in metalloprotease activities associated with age were most striking in the lateral lobe and included activities of 52, 55, 81, 93, 113, and 117 kDa at 18 months of age. Castration for 30 days at this age resulted in a decline in the 52, 55, 113, and 117 kDa activities and an increase in activities of the 70, 81, and 93 kDa forms. These latter metalloprotease activities were also increased in the dorsal lobe after castration. Our results suggest that some metalloprotease activities increased in the dorsal lobe after castration. Our results suggest that some metalloprotease activities increased in the lateral lobe with age possibly result from an increased accumulation of secretory proteins (i.e., 52, 55, 113, and 117 kDa), whereas the 70, 81, and 93 kDa metalloprotease activities may be related to possible prostatitis and/or involved in changes in tissue organization. The increased expression of metalloprotease activities in the lateral and dorsal prostate lobes with aging, and castration upon aging, may be indicative of altered hormonal regulation of these proteases in these lobes.

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.

Plasminogen activator activities in the ventral and dorsolateral prostatic lobes of aging Fischer 344 rats.

Plasminogen activator (PA) activities were measured in extracts of ventral and dorsolateral prostate lobes in Fischer 344 (F344) rats of different ages. Ventral prostates of 30-month-old animals demonstrated widespread epithelial atrophy, some foci of inflammation, intraglandular atypical hyperplasia and/or adenocarcinoma, and a marked increase in PA activities compared with younger animals. Both low- and high-molecular-weight forms of activator were found in zymograms and both forms of activator showed an increase at 30 months. However, the PA activity of the aged rats showed a greater resistance to amiloride inhibition, suggesting a greater relative increase in the tissue-type activator activity than that of urokinase. These changes were not observed in the dorsolateral prostate. The lobe-specific changes in activator activity appear related to dysplasia/neoplasia and not aging.

Papilloma and carcinoma production in DMBA-initiated, onion oil-promoted mouse skin.

Groups of 20 females Ha/ICR mice were initiated with 25 micrograms 7,12-dimethylbenz[a]anthracene (DMBA) and promoted one week later with topical treatments three times per week of 5 micrograms phorbol myristate acetate (PMA) and/or onion oil or garlic oil. Promotion was continued for 49 weeks in most experiments. Promotion was continued for 60 weeks in the experiment that evaluated the effect of time intervals between PMA and garlic oil. All experiments were conducted with 0.2 ml acetone solutions of agents. Onion oil, but not garlic oil, was a weak promoter in mouse skin. A 1-mg dose produced five papillomas in three mice and one carcinoma in 330 days (18 survivors). The 10-mg dose was more effective; it produced cumulative yields of 56 papillomas in 14 mice and 7 carcinomas in 4 mice in 345 days (14 survivors). Onion oil is neither an initiator nor a whole carcinogen. The effects of intervals between PMA and a 1-mg dose of onion or garlic oil were determined. These intervals were -2 hrs, -1 hr, -0.5 hr, +0.5 hr, +1 hr, and +2 hrs with respect to time of PMA application. Maximal inhibition of papillomas by onion oil was observed at the +0.5-hr interval and was similar to that previously reported. Garlic oil is not a promoter. It inhibited papillomas at the +0.5-hr, +1.0-hr, and +2.0-hr intervals but did not appear to affect carcinoma production.

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.

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.

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.

Plasminogen activator activities in the developing rat prostate.

Plasminogen activator (PA) activities were measured in the rat prostatic complex and individual prostatic lobes during early postnatal and pubertal development and in sexually mature adult rats. There was no significant change in PA activity during postnatal prostate development. However, during sexual maturation with puberty, there was a decline in PA activity in the ventral (3-fold), dorsolateral (22-fold), and anterior (19-fold) prostate lobes when activity was expressed per unit protein. A decrease in activity of 25- and 11-fold was found for the dorsolateral and anterior lobes, respectively, when activity was expressed per unit DNA. There was no change in activity in the ventral lobe. The adult ventral prostate (and its secretion) have 3 broad bands of low molecular mass (approximately 23 and 26-32 kDa) plasminogen-independent protease activities. Proteases of these molecular sizes as well as an activity of 170 kDa were detected in the dorsolateral prostate. The former proteases in the ventral and dorsolateral lobes were first found at 21 days of age, whereas the 170 kDa protease was found in dorsolateral prostate immediately post-puberty (48 days). The low molecular mass plasminogen-independent proteases were also able to activate plasminogen (determined by zymography) and hence contribute to the total measured PA activity. Thus, at 21 days of age, the specific activity of plasminogen-dependent protease declined, since the total measured PA-specific activity did not change. Plasminogen-dependent activities in ventral, dorsolateral, and anterior prostate lobes of adult rats were found as doublets of approximately 57-59 kDa and 36-38 kDa.(ABSTRACT TRUNCATED AT 250 WORDS)

Urokinase-type plasminogen activator in alveolar macrophages and bronchoalveolar lavage fluid from normal and smoke-exposed hamsters and humans.

Our purpose was to identify and characterize plasminogen activator (PA) activity in alveolar macrophages (AMs) and lower respiratory tract fluid from hamsters and humans. Freshly harvested hamster AMs had barely detectable PA intracellularly and no detectable activity in concentrated lavage fluid. PA secretion was detected in conditioned media of hamster AMs. Secretion was enhanced sixfold by phorbol myristate acetate (PMA) and suppressed by dexamethasone. In contrast to hamsters, PA activity was readily detected in lysates of freshly harvested human AMs and lavage fluid. However, secretion of PA by human AMs was not detected, and neither PMA nor dexamethasone affected PA activity. PA from hamster or human AMs has a urokinase-type enzyme with molecular weights of 49,000 or 55,000, respectively. Cigarette smoking did not alter PA activity by hamster or human AMs.

Induction and desensitization of plasminogen activator gene expression by tumor promoters.

Tumor promoting phorbol esters and mezerein strongly induced plasminogen activator (urokinase, uPA) synthesis in porcine kidney cell cultures (LLC-PK1). Induction was due to increased uPA-mRNA levels which rose from 10 to 300 molecules/cell within 2 h of exposure to 16 nM phorbol myristate acetate. We have compared the action of tumor promoters with that of 8-bromo-cAMP, another potent inducer of uPA; the similarities between the two kinds of induction were: both involved transcriptional activation of the uPA gene; both were rapid in onset, changes in transcription rate being detectable within 10-20 min; the initial rates of transcription and uPA-mRNA accumulation were substantial and in the same order of magnitude; neither class of inducer required protein synthesis to stimulate uPA transcription. The main contrast between the two types of agents was that the uPA response to tumor promoters was transient whereas that to cAMP compounds was sustained: cultures rapidly lost their response to tumor promoters within 2 h after initial exposure while retaining responsiveness to cAMP-related agents. The cells developed a specific drug-induced desensitization which was slowly reversed after tumor promoters were removed from the culture medium. Since protein kinase C is now well established as the receptor for phorbol-derived and several other tumor promoters it will be of interest to determine whether desensitization occurs at the level of receptor.

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.