Towards area wide management of insect vectored viruses of tomatoes in the Bowen district.

The Bowen region of Northern Queensland is an important winter production area for tomatoes in Australia. There are three economically important viruses in the region that affect tomato, Tomato yellow leaf curl virus (TYLCV), Tomato spotted wilt virus (TSWV) and Potato leafroll virus (PLRV), which are vectored by whiteflies, thrips and aphids, respectively. An area wide management approach is required to lower the primary inoculum throughout the district. To this end, we undertook investigations into the virus incidence and alternative hosts for the virus and vectors in different cropping regions throughout the district, as well as local management options such as insecticide application and possible non-host cover crops for the wet-season break in production. The initial incidence of Potato leafroll virus was very high, most probably due to abnormal weather patterns for the district, and has ceased to be a problem. Tomato yellow leaf curl virus is a continual problem even at the beginning of the season, indicating large reservoir host(s) in the environment. Only four alternative hosts have been identified: Stachytarpheta jamaicensis (TSWV), Solanum americanum (PLRV and TYLCV) Trianthema portulacastrum (TYLCV), and Amaranthus viridis(TLYCV). Different insecticide and application options were trialled for protection against Tomato yellow leaf curl virus, with the best possible option yielding marketable fruit more than ninety percent of a resistant hybrid. A trial of yield vs time of infection of TYLCV found that whitefly exclusion for 6 weeks post-transplant yielded an average increase of nearly three kilograms of marketable fruit per plant. A number of pulse crops have been confirmed as non-hosts of tomato yellow leaf curl for use as cover crops in the wet-season break. Most of the production has moved to dual resistant TYLCV/TSWV hybrids, though an area wide management program still needs to be established to reduce the primary inoculum throughout the district, giving growers more varietal options, especially early in the season.

Abnormal brain structure in youth who commit homicide.

BACKGROUND: Violence that leads to homicide results in an extreme financial and emotional burden on society. Juveniles who commit homicide are often tried in adult court and typically spend the majority of their lives in prison. Despite the enormous costs associated with homicidal behavior, there have been no serious neuroscientific studies examining youth who commit homicide. METHODS: Here we use neuroimaging and voxel-based morphometry to examine brain gray matter in incarcerated male adolescents who committed homicide (n = 20) compared with incarcerated offenders who did not commit homicide (n = 135). Two additional control groups were used to understand further the nature of gray matter differences: incarcerated offenders who did not commit homicide matched on important demographic and psychometric variables (n = 20) and healthy participants from the community (n = 21). RESULTS: Compared with incarcerated adolescents who did not commit homicide (n = 135), incarcerated homicide offenders had reduced gray matter volumes in the medial and lateral temporal lobes, including the hippocampus and posterior insula. Feature selection and support vector machine learning classified offenders into the homicide and non-homicide groups with 81% overall accuracy. CONCLUSIONS: Our results indicate that brain structural differences may help identify those at the highest risk for committing serious violent offenses.

Transplacental antioxidants inhibit lung tumors in mice exposed to cigarette smoke after birth: a novel preventative strategy?

Birth is characterized by an intense oxidative stress resulting in nucleotide alterations and gene overexpression in mouse lung. We showed that cigarette smoke (CS) is carcinogenic when exposure starts soon after birth and applied this bioassay to evaluate the efficacy of chemopreventive agents. The present study evaluated whether administration of the antioxidants N-acetyl-L-cysteine (NAC) and vitamin C or ascorbic acid (AsA) during pregnancy can protect strain H Swiss mice exposed to CS after birth. Exposure to CS, for 4 months, of newborns from untreated mice resulted in significant alterations at 8 months of life, including alveolar epithelial hyperplasia, emphysema, blood vessel proliferation, microadenomas, adenomas, and malignant tumors in lung, liver parenchymal degeneration and urinary bladder epithelium hyperplasia. Treatment throughout pregnancy with either NAC, a scavenger of reactive oxygen species, or AsA, an electron donor, did not affect fertility, parity, and body weight of newborns. Prenatal antioxidants significantly inhibited most lesions in adult mice exposed to CS since birth. For instance, the incidence of emphysema was reduced from 27.5% in CS-exposed mice that were untreated during pregnancy to 7.1% and 14.0% in those treated prenatally with NAC and AsA, respectively. Lung adenomas were reduced from 34.8% to 16.7% and 9.3%, respectively. Malignant lung tumors were reduced from 13.0% to 4.7% by prenatal AsA. Liver parenchymal degeneration was reduced from 58.0% to 14.3% by prenatal NAC. These data mechanistically support a "transplacental chemoprevention" strategy, aimed at protecting the newborn from oxidative stress and the adult from CS-related diseases appearing later in life.

Identification of viral and non-viral reverse transcribing elements in pineapple (Ananas comosus), including members of two new badnavirus species.

A previously published partial sequence of pineapple bacilliform virus was shown to be from a retrotransposon (family Metaviridae) and not from a badnavirus as previously thought. Two newly discovered sequence groups isolated from pineapple were associated with bacilliform virions and were transmitted by mealybugs. Phylogenetic analyses indicated that they were members of new badnavirus species. A third caulimovirid sequence was also amplified from pineapple, but available evidence suggests that this DNA is not encapsidated, but more likely derived from an endogenous virus.

Pouch young removal and return to oestrus in wild southern hairy-nosed wombats (Lasiorhinus latifrons).

The southern hairy-nosed wombat (Lasiorhinus latifrons) is a seasonal breeding, burrowing marsupial adapted to a semi-arid environment and the closest relative of the endangered northern hairy-nosed wombat (Lasiorhinus krefftii). Females typically give birth to one to two young every 3 years with young weaned at 360-400 days. This study examined the occurrence of polyoestry in a wild population of southern hairy-nosed wombats, and in particular the ability of this species to produce additional offspring in the same breeding season if a young was prematurely lost or removed. Pouch young were removed during the breeding seasons of 1996/1997 and 2003. No females from the 1996 (n=3)/1997 (n=3) group gave birth to a second pouch young in the same breeding season. However, two females in this group gave birth to young the following season. In contrast, all the 2003 group of females (n=6) produced a second offspring in the same breeding season after removal of pouch young (RPY). The reason for the different response to RPY between the two groups is unknown. These studies confirm that southern hairy-nosed wombats are polyoestrus in the wild and are capable of producing more than one offspring in a single breeding season. Females that failed to return to oestrus in the breeding season that pouch young were removed bred again in the following season. Rapid replacement of southern hairy-nosed wombat pouch young in the same breeding season as RPY suggests that this procedure, linked to either hand-rearing or interspecific cross-fostering, should be seriously considered as a priority conservation action to increase the population size of the critically endangered sister species, the northern hairy-nosed wombat.

Prostate intraepithelial neoplasia in Noble rats, a potential intermediate endpoint for chemoprevention studies.

In most prostate chemoprevention studies conducted with animal models, the incidence and multiplicity of tumours have been used as endpoints for efficacy. However, the latency of tumours is usually over 1 year, making these studies costly and time consuming. The main purpose of this study was to assess the utility of prostate intraepithelial neoplasia (PIN), induced in Noble rats by continuous testosterone + oestradiol (T + E) administration, as a potential intermediate endpoint biomarker of efficacy in chemoprevention studies. Noble rats at the age of 12 weeks were treated for 36 weeks with T + E given subcutaneously via Silastic capsules. The incidence and multiplicity of PIN were assessed in various prostate glands by serial sections generated at three separate tissue levels. The efficacy of dehydroepiandrosterone (DHEA) and DHEA 8354 (1000 and 2000 mg/kg diet), difluoromethylornithine (DFMO) (1000 and 2000 mg/kg diet) and oltipraz (125 and 250 mg/kg diet) to inhibit PIN was assessed in two independent sets of experiments. T + E induced multiple PIN in the dorsolateral prostate (DLP) of 80-100% of the animals. DHEA and DHEA 8354 did not affect the incidence but decreased the multiplicity of PIN in the DLP, from 3.2 +/- 1.0 in control group to 1.5 +/- 1.0 in the low-dose and to 1.6 +/- 0.6 in the high-dose group for DHEA (P<0.05 and P<0.02, respectively), and to 1.9 +/- 0.8 in the high-dose (P<0.05) DHEA 8354. Both agents did not affect PIN in anterior prostate, seminal vesicles or ventral prostate. In a second experiment, DFMO and oltipraz were found not effective in inhibiting PIN. In this study, we provide new evidence that PIN in Noble rats, induced by continuous T + E treatment, is a useful intermediate endpoint for determining the efficacy of DHEA and other potential chemopreventive agents. The hormonal pathogenesis, high multiplicity, short latency, preferential location in the DLP, similarity in morphology and biology to PIN of human prostate, and the sensitivity to agents that suppress prostate carcinogenesis, makes PIN in Noble rats a promising intermediate endpoint for chemoprevention studies.

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.

Chemopreventive agents induce a senescence-like phenotype in rat mammary tumours.

Terminal replicative senescence (TRS) is a physiological process associated with terminal differentiation, shortening of the telomere, and lack of proliferative activity. Immortalised and tumour cells have lost their differentiation potential and the ability to develop a senescence phenotype. Recently, others and we [11] have observed that some antitumour agents and radiation induce a senescence-like phenotype (SLP) in human immortalized and tumour cell lines. The main purpose of this study was to identify senescence-like cells (SLC) in mammary tumours of rats and assess whether chemopreventive agents that have been used for the prevention and/or treatment of breast cancer can induce a SLP in tumour cells. Sprague-Dawley rats with N-methyl-N-nitrosourea (MNU)-induced mammary tumours were randomised and treated with tamoxifen, vorozole, 4-(hydroxyphenyl)retinamide (4-HPR), or 9-cis-retinoic acid (9cRA). The SLC in mammary tumours were identified and characterised by: (a) SA-beta-Gal staining method, which has been considered specific for human cells in TRS (b) staining for lipofuscin, which, although not specific, accumulates in the cytoplasm of cells in senescence; (c) lack of 5-Bromodeoxyuridine (BrdU) labelling after continuous (7 days) infusion of BrdU via osmotic pumps; (d) 90 degrees side light scatter (9OLS) as evaluated by flow cytometry; and (e) decreased telomerase activity. We found that in control tumours, SA-beta-Gal-positive cells were rare (below 1.0%) among the tumour cells, stroma fibroblast, myoepithelial and endothelial cells. SA-beta-Gal-positive cells increased significantly in the tumours treated with chemopreventive agents and this was associated with a lack of proliferative activity, increased cell granularity, lipofuscin accumulation, and decreased telomerase activity. Thus, in this study we provide for the first time evidence that cells in replicative senescence are present in mammary tumours of rats and that chemopreventive agents can suppress tumor growth by a novel cellular mechanism, inducing a SLP in the tumor cells.

Effects of dietary N-(4-hydroxyphenyl)retinamide on N-nitrosomethylbenzylamine metabolism and esophageal tumorigenesis in the Fischer 344 rat.

BACKGROUND: 9-cis-Retinoic acid (9-cis-RA) and N-(4-hydroxyphenyl)retinamide (4-HPR) are effective chemopreventive agents against epithelial tumors in the oral cavity, breast, and prostate. We tested the inhibitory activity of these retinoids against N-nitrosomethylbenzylamine (NMBA)-induced tumorigenesis in the rat esophagus. METHODS: Male Fischer 344 rats were randomly assigned to receive diets either lacking or containing 9-cis-RA or 4-HPR for 1 week before tumor initiation with NMBA and then for the duration of the study. NMBA metabolism, O(6)-methylguanine adduct formation, and cytochrome P450 messenger RNA (mRNA) expression in the esophagi of the rats were studied to investigate the mechanisms by which dietary 4-HPR affects tumorigenesis. All statistical tests were two-sided. RESULTS: Dietary 4-HPR resulted in a dose-dependent and statistically significant enhancement (P<.05) of tumorigenesis in response to NMBA. In two different tumor bioassays, the mean tumor multiplicity for rats fed the highest concentration of dietary 4-HPR (0.8 g/kg diet) was increased by 5.9 tumors (95% confidence interval [CI] = 1.7 to 10.1 tumors) and 6.7 tumors (95% CI = 5.6 to 7.8 tumors) compared with the mean tumor multiplicity for rats that received the control diet lacking 4-HPR. Animals fed diets containing 9-cis-RA displayed no statistically significant increase in tumorigenesis. Compared with animals fed a diet lacking 4-HPR, animals fed 4-HPR had increased NMBA metabolism in esophageal explant cultures and had higher levels of O(6)-methylguanine DNA adducts and CYP2A3 mRNA in their esophagi. CONCLUSIONS: Dietary 4-HPR enhances tumorigenesis in response to NMBA in the rat esophagus by increasing tumor initiation events. Dietary 4-HPR may exert paradoxical effects at some sites, such as the aerodigestive tract, by modulating the bioactivation of carcinogens in target tissues.

Chemoprevention by difluoromethylornithine: correlation of an in vitro human cell assay with human clinical data for biomarker modulation.

The efficacy of difluoromethylornithine (DFMO) as a chemopreventive agent has been tested in vitro using a human epidermal cell (HEC) assay with growth inhibition and involucrin induction as endpoints. Suppression of polyamine content is currently being utilized as a biomarker in clinical trials for the chemopreventive efficacy of DFMO against colon cancer formation. We have now examined the effects of DFMO on suppression of polyamine content in the HEC assay. The findings indicate 1) the % change in spermidine to spermine ratio and the depletion of putrescine show excellent correlation with chemopreventive efficacy in vitro; 2) the effective concentrations in vitro overlap the plasma concentrations in the clinical trial. These observations serve as further validation of the usefulness of the HEC assay as a screen for chemopreventive efficacy.

Identification of retinamides that are more potent than N-(4-hydroxyphenyl)retinamide in inhibiting growth and inducing apoptosis of human head and neck and lung cancer cells.

The synthetic retinoid, N-(4-hydroxyphenyl)retinamide (4HPR), which is currently being evaluated in clinical trials for cancer prevention and therapy, inhibits the growth of a variety of malignant cells through induction of apoptosis. However, in the majority of tumor cells, this inhibitory effect of 4HPR requires high concentrations (>1 microM), which exceed the peak plasma level measured in humans. In the present study, we compared and contrasted the effects of several synthetic retinamides on the growth of human lung and head and neck cancer cells in vitro. We found that some retinamides, especially N-(2-carboxyphenyl)retinamide (2CPR), exhibited better growth inhibitory effects than 4HPR in some of the cell lines. 2CPR exerted potent growth inhibitory effects in 5 of 10 head and neck cancer cell lines and in 1 of 10 lung cancer cell lines (IC(50), <0.8 microM). 2CPR (1 microM) induced apoptosis ranging from 10 to 60% in four of five cell lines, whereas 4HPR was ineffective at the same concentration. Unlike 4HPR, 2CPR (up to 10 microM) failed to induce reactive oxygen species production in these sensitive cell lines but could activate caspases 3 and 7 as well as increase poly(ADP-ribose)polymerase cleavage. Interestingly, the effect of 2CPR on cell growth could be suppressed by the specific retinoic acid receptor pan antagonist AGN193109. Our results suggest that 2CPR acts via retinoic acid receptors and may be a good candidate for prevention and treatment of some head and neck and lung cancers.

Effects of novel phenylretinamides on cell growth and apoptosis in bladder cancer.

Superficial bladder cancer is a major target for chemoprevention. Retinoids are important modulators of epithelial differentiation and proliferation and are effective in the treatment and prevention of several epithelial cancers. One class of compounds, the retinamides, is structurally similar to other retinoids but have the added feature of being potent apoptosis inducers. Among these, fenretinide (N-[4-hydroxyphenyl]retinamide), or 4HPR, has promise for bladder cancer chemoprevention and is currently under Phase III study in this setting. In addition to 4HPR, there are several new structurally related phenylretinamides bearing hydroxyl, carboxyl, or methoxyl residues on carbons 2, 3, and 4 of the terminal phenylamine ring [designated N-(2-hydroxyphenyl)retinamide, N-(3-hydroxyphenyl)retin amide, N-(2-carboxyphenyl)retin- amide, N-(3-carboxyphenyl)retin amide, N-(4-carboxy- phenyl)retinamide, and N-(4-methoxyphenyl)retinamide, respectively]. The objective of this study was to compare the growth inhibitory and apoptotic effects of these phenylretinamides with 4HPR in human bladder transitional cell cancer-derived cell lines of varying histological grade (RT4, grade 1; UM-UC9 and UM-UC10, grade 3; and UM-UC14, grade 4) by cell counting, cell cycle fluorescence-activated cell sorter analysis and a dual stain apoptosis assay. All of the seven phenylretinamides reduced cell number, altered the cell cycle distribution, and induced apoptosis when administered at a concentration of 10 microM, which is within the pharmacologically achievable range. Although the relative potencies of the phenylretinamides varied depending on the cell line, N-(3-hydroxy phenyl)retin- amide was the most active with significantly greater growth inhibition than 4HPR in all of the four cell lines. These in vitro findings warrant further study of these novel phenylretinamides, which may have potential as preventive or therapeutic agents in transitional cell cancer.

Prefrontal cortical sulcal widening associated with poor treatment response to clozapine.

Increased sulcal widening in the prefrontal cortex of patients with schizophrenia may be associated with a poor treatment response to clozapine. To further evaluate this, we examined data from patients treated with clozapine in our center. Patients with the greatest degree of improvement (n=26) and those with no improvement (n=10) were compared. Computerized tomography (CT) scans were rated blindly on a visual scale of prefrontal sulcal widening. Patients with the greatest degree of functional improvement had significantly less prefrontal sulcal widening than those whose symptoms remained unchanged. There was no relationship between clozapine response and general sulcal widening. These data support the link between the superior therapeutic efficacy of clozapine and the integrity of the prefrontal cortex.

Agents, biomarkers, and cohorts for chemopreventive agent development in prostate cancer.

Chemoprevention is the use of agents to slow progression of, reverse, or inhibit carcinogenesis thereby lowering the risk of developing invasive or clinically significant disease. With its long latency, high incidence and significant morbidity and mortality, prostate cancer is a relevant target for chemoprevention. Developing rational chemopreventive strategies for prostate cancer requires well-characterized agents, suitable cohorts, and reliable intermediate biomarkers of cancer. Chemopreventive agent requirements are experimental or epidemiologic data showing efficacy, safety on chronic administration, and a mechanistic rationale for activity. Current promising agents include antiandrogens and antiestrogens; steroid aromatase inhibitors; retinoids and their modulators; 5alpha-reductase inhibitors; vitamins D, E, and analogs; selenium compounds; carotenoids; soy isoflavones; dehydroepiandrostenedione and analogs; 2-difluoromethylornithine; lipoxygenase inhibitors; apoptosis inducers; and nonsteroidal anti-inflammatory drugs. Identifying biomarkers and validating them as surrogate endpoints for cancer incidence are critical for prostate chemoprevention trials. Potentially useful biomarkers for prostate chemoprevention are associated with histologic, proliferative, differentiation-related, biochemical, and genetic/regulatory features of prostatic disease. In that the prostate is not easily visualized, critical issues also include adequacy and consistency of tissue sampling. Various drugs for the chemoprevention of prostate cancer are now under evaluation in phase 1, 2, and 3 clinical trials. Cohort selection should be based on various patient characteristics (stage of the disease, previous cancers or premalignant lesions, or high risk factors) and should be conducted within the context of standard treatment.

Effects of acute and chronic body weight gain reductions in the evaluation of agents for efficacy in mammary cancer prevention.

Studies were performed to determine the effects of moderate decreases in body weight gain on mammary carcinogenesis. The levels of depressions in weight gain were those often observed in the evaluation of chemopreventive agents. In the first experiment, the effects of acute and chronic reductions of body weight gain when started after carcinogen treatment were examined in young rats (MNU at 50 days of age). Significant decreases (36%) in mammary cancers occurred in groups of rats that underwent a 12% acute reduction in body weight gain as compared with ad libitum controls. In contrast, chronic weight reductions of up to 12% had minimal effects on cancer multiplicities, while a 15% chronic reduction significantly decreased cancer numbers (26%). A second experiment evaluated the efficacy of toremifene (7.0 mg/kg diet), an estrogen/anti-estrogen, and the effect of toremifene-matched body weight gain reduction that occurred during the study. Toremifene caused a chronic reduction in body weight that resulted in a 10% decrease in final body weight at the end of the study. While toremifene-treated rats exhibited a 67% decrease in the number of mammary cancers, the rats which similarly exhibited a 10% decrease in final body weight showed only a 14% decrease in cancer number. Thus, the weight effects observed with toremifene, similar estrogens/anti-estrogens, and other classes of chemopreventive compounds (where chronic body weight reductions are 10% or less) imply that the body weight reduction has a limited effect on overall chemopreventive activity. A third study examined the effect of chronic body weight gain reduction on mammary cancers induced in older rats (MNU given at 100 days of age). This model more closely resembles the status of the breast tissue of mature women currently enrolled in clinical trials of chemopreventive agents. Under these conditions chronic reductions in body weight up to 15% had minimal effects on mammary carcinogenesis. These data further demonstrated that acute body weight reductions in young rats at the time of carcinogen treatment can be a concern in interpretation of the chemopreventive activity of an agent, but that moderate chronic depressions of body weight gain probably do not play a significant role.

A quantitative angiogenesis model for efficacy testing of chemopreventive agents.

One of the approaches in chemoprevention to prevent or delay the progression of precancerous lesions, is to apply chemopreventive agents that can potentially block angiogenesis. A quantitative in vivo angiogenesis inhibition assay was developed to test the efficacy of twelve chemopreventive agents that represent different chemical classes and multiple biological activities, using the chick chorioallantoic membrane (CAM) model and an oncogene-transfected angiogenic cell line (6 Ti ras/SV myc # 4). These tumorigenic cells held by a primary agarose pellet, were placed alone or with a secondary pellet incorporating five concentrations of the test agent, on an exposed CAM of 7-day-old chick embryo for 72 hours in a humidified chamber at 35 degrees C. The cell-induced angiogenic blood vessels, including the microvessels radiating from the cell pellet focal area, were scored using a computerized custom image analysis system. The results show that nonsteroidal antiinflammatory drugs (NSAIDS); aspirin, sulindac, sulindac sulfide and sulindac sulfone, were effective inhibitors of cell-induced angiogenesis (23-66%). Aspirin displayed a dose-dependent response with the highest inhibition at 300 microM and an EC50 (the effective molar concentration that inhibits angiogenesis by 50%) of 26 microM. Sulindac sulfone was more effective than sulindac with an EC50 of 5 microM versus 85 microM. However, sulindac sulfide showed an intermediate response with an EC50 of 41 microM. The retinoids; all-trans-retinoic acid (ATRA), 9-cis-retinoic acid (9-cis-RA), and 13-cis-retinoic acid (13-cis-RA) were also highly effective inhibitors of cell-mediated CAM-angiogenesis. 13-cis-RA with an EC50 of 3.6 nM, has been the most efficacious test agent. > 400-fold more effective than 9-cis-RA (1.5 microM). ATRA exhibited an intermediate response between 9-cis-RA and 13-cis-RA with an EC50 of 0.3 microM, and was 100-fold more efficacious than 9-cis-RA. However, the synthetic retinoid, N-(4-hydroxyphenyl) retinamide (4-HPR), was not an effective inhibitor of CAM angiogenesis. Thalidomide, a compound with multiple biological activities, exhibited dose-dependent inhibition ranging from 10-1000 microM with an EC50 of 19 microM. Other agents that exhibited dose-dependent inhibition included Bowman-Birk inhibitor (BBI), EC50: 10 microg/ml, tamoxifen, EC50, 0.05 microM and difluoromethyl omithine (DFMO), with an EC50 of 13 microM. These results suggest that tumor-associated angiogenesis can be modulated by non-toxic concentrations of chemopreventive agents representing multiple biological activities and multiple targets.

Effect of the aromatase inhibitor vorozole on estrogen and progesterone receptor content of rat mammary carcinomas induced by 1-methyl-1-nitrosourea.

Vorozole, a nonsteroidal aromatase inhibitor, impedes the post-initiation stage of chemically induced mammary carcinogenesis. While various aspects of vorozole's effects on mammary carcinoma development have been investigated, little attention has been directed to determining the estrogen receptor (ER) and progesterone receptor (PR) content of mammary carcinomas that arise despite vorozole treatment. Female Sprague-Dawley rats were given an i.p. injection of 50mg MNU/kg body weight at 21 days of age and placed on diet supplemented with 0 or 3 mg vorozole/kg, which had no effect on mammary tumor development. Histologically confirmed carcinomas were evaluated for ER and PR by immunohistochemistry. In the control group, 78.8% of carcinomas were ER positive with an ER content ranging from 13.8 to 40.0%, similar to ER content of mammary ductal epithelial cells from non-carcinogen treated animals. PR content ranged from 4.4 to 45.2% and also was similar to levels of PR observed in ductal epithelial cells. ER was not correlated with PR in mammary carcinomas (r = 0.05, p > 0.80), whereas there was a significant correlation in ductal epithelium (r = 0.86, p = 0.006). In vorozole-treated rats, no ER negative carcinomas were observed and overall ER expression by vorozole was elevated (p < 0.03). All carcinomas from vorozole-treated rats expressed PR (2.5-60.2%) and correlation between ER and PR content was numerically greater in carcinomas from vorozole-treated animals (r = 0.42, p = 0.09). These data, which are considered hypothesis generating, provide evidence that low doses of vorozole in the diet select for mammary carcinomas with an increased ER positive phenotype.

Comparative tissue-specific toxicities of 20 cancer preventive agents using cultured cells from 8 different normal human epithelia.

Comparative toxicity was determined for twenty potential chemopreventive agents in the Human Epithelial Cell Cytotoxicity (HECC) Assay using epithelial cell cultures from eight different tissues including: skin, kidney, breast, bronchus, cervix, prostate, oral cavity, and liver. The endpoints assessed were inhibition of: growth at 3 and 5 days; mitochondrial function; and proliferating cell nuclear antigen or albumin expression. Difluoromethylornithine (DFMO), s-allylcysteine, dehydroepiandrosterone (DHEA) analogue 8543, l-selenomethionine, and vitamin E acetate were not toxic or only produced mild toxicity with all endpoints in all eight cell types. N-acetyl-l-cysteine, calcium chloride, DHEA, genistein, ibuprofen, indole-3-carbinol, 4-hydroxyphenylretinamide (4-HPR), oltipraz, piroxicam, phenylethyl isothiocyanate, 9-cis-retinoic acid, and p-xylylselenocyanate each showed at least a 10-fold decrease in their TC(50) (toxic concentration that inhibited growth by 50%) for at least one endpoint with one or more cell types. For some agents such as DHEA and piroxicam, the TC(50)s for growth inhibition were 10-fold lower after 5 days compared with 3 days. Unique tissue-specific toxicity was observed for each toxic agent suggesting that tissue-specific effects are the rule rather than the exception. The HECC Assay is effective in identifying tissue-specific toxicity for chemopreventive agents and may help to identify potential toxicity problems in phase I human clinical trials.

Modulation of Ki67, p53 and RARbeta expression in normal, premalignant and malignant human oral epithelial cells by chemopreventive agents.

BACKGROUND: Aberrant expression of Ki67, p53 and RARbeta are characteristic of many tumor types including those of the oral cavity. Chemopreventive agents may act by modulating their expression to more normal levels. MATERIALS AND METHODS: The effects of 21 chemopreventive agents on the expression of Ki67, p53 and RARbeta were determined using a human in vitro model of normal, premalignant and malignant oral epithelial cell lines. RESULTS: Ki67 and mutant p53 (mtp53) were overexpressed in both the premalignant and malignant cell lines, whereas expression of RARbeta was high in the normal, low in the premalignant and not detectable in the malignant cell lines. Most of the agents selectively inhibited the expression of Ki67 in the premalignant and malignant cell lines. Eight of the 21 agents increased, while four agents decreased, the levels of mtp53 protein in the premalignant cell line. In the malignant cell line, five of the agents increased, while ten agents decreased mtp53 protein levels. The agents increased RARbeta expression to near normal levels in the premalignant cell line. CONCLUSION: The data suggest that the suppression of Ki67 and mtp53 are good indicators of the effectiveness of agents in premalignant and malignant oral cells, whereas the enhancement of RARbeta is a measure of effectiveness in premalignant oral cells.