Inhibition of farnesyltransferase induces regression of mammary and salivary carcinomas in ras transgenic mice.

For Ras oncoproteins to transform mammalian cells, they must be post-translationally modified with a farnesyl group in a reaction catalysed by the enzyme farnesyl-protein transferase (FPTase). Inhibitors of FPTase have therefore been proposed as anti-cancer agents. We show that L-744,832, which mimics the CaaX motif to which the farnesyl group is added, is a potent and selective inhibitor of FPTase. In MMTV-v-Ha-ras mice bearing palpable tumours, daily administration of L-744,832 caused tumour regression. Following cessation of treatment, tumours reappeared, the majority of which regressed upon retreatment. No systemic toxicity was found upon necropsy of L-744,832-treated mice. This first demonstration of anti-FPTase-mediated tumour regression suggests that FPTase inhibitors may be safe and effective anti-tumour agents in some cancers.

A farnesyltransferase inhibitor induces tumor regression in transgenic mice harboring multiple oncogenic mutations by mediating alterations in both cell cycle control and apoptosis.

The farnesyltransferase inhibitor L-744,832 selectively blocks the transformed phenotype of cultured cells expressing a mutated H-ras gene and induces dramatic regression of mammary and salivary carcinomas in mouse mammary tumor virus (MMTV)-v-Ha-ras transgenic mice. To better understand how the farnesyltransferase inhibitors might be used in the treatment of human tumors, we have further explored the mechanisms by which L-744,832 induces tumor regression in a variety of transgenic mouse tumor models. We assessed whether L-744,832 induces apoptosis or alterations in cell cycle distribution and found that the tumor regression in MMTV-v-Ha-ras mice could be attributed entirely to elevation of apoptosis levels. In contrast, treatment with doxorubicin, which induces apoptosis in many tumor types, had a minimal effect on apoptosis in these tumors and resulted in a less dramatic tumor response. To determine whether functional p53 is required for L-744,832-induced apoptosis and the resultant tumor regression, MMTV-v-Ha-ras mice were interbred with p53(-/-) mice. Tumors in ras/p53(-/-) mice treated with L-744,832 regressed as efficiently as MMTV-v-Ha-ras tumors, although this response was found to be mediated by both the induction of apoptosis and an increase in G1 with a corresponding decrease in the S-phase fraction. MMTV-v-Ha-ras mice were also interbred with MMTV-c-myc mice to determine whether ras/myc tumors, which possess high levels of spontaneous apoptosis, have the potential to regress through a further increase in apoptosis levels. The ras/myc tumors were found to respond nearly as efficiently to L-744,832 treatment as the MMTV-v-Ha-ras tumors, although no induction of apoptosis was observed. Rather, the tumor regression in the ras/myc mice was found to be mediated by a large reduction in the S-phase fraction. In contrast, treatment of transgenic mice harboring an activated MMTV-c-neu gene did not result in tumor regression. These results demonstrate that a farnesyltransferase inhibitor can induce regression of v-Ha-ras-bearing tumors by multiple mechanisms, including the activation of a suppressed apoptotic pathway, which is largely p53 independent, or by cell cycle alterations, depending upon the presence of various other oncogenic genetic alterations.

Mouse mammary tumor virus-Ki-rasB transgenic mice develop mammary carcinomas that can be growth-inhibited by a farnesyl:protein transferase inhibitor.

For Ras oncoproteins to transform mammalian cells, they must be posttranslationally modified with a farnesyl group in a reaction catalyzed by the enzyme farnesyl:protein transferase (FPTase). Inhibitors of FPTase have therefore been developed as potential anticancer agents. These compounds reverse many of the malignant phenotypes of Ras-transformed cells in culture and inhibit the growth of tumor xenografts in nude mice. Furthermore, the FPTase inhibitor (FTI) L-744,832 causes tumor regression in mouse mammary tumor virus (MMTV)-v-Ha-ras transgenic mice and tumor stasis in MMTV-N-ras mice. Although these data support the further development of FTIs, it should be noted that Ki-ras is the ras gene most frequently mutated in human cancers. Moreover, Ki-RasB binds more tightly to FPTase than either Ha- or N-Ras, and thus higher concentrations of FTIs that are competitive with the protein substrate may be required to inhibit Ki-Ras processing. Given the unique biochemical and biological features of Ki-RasB, it is important to evaluate the efficacy of FTIs or any other modulator of oncogenic Ras function in model systems expressing this Ras oncoprotein. We have developed strains of transgenic mice carrying the human Ki-rasB cDNA with an activating mutation (G12V) under the control of the MMTV enhancer/promoter. The predominant pathological feature that develops in these mice is the stochastic appearance of mammary adenocarcinomas. High levels of the Ki-rasB transgene RNA are detected in these tumors. Treatment of MMTV-Ki-rasB mice with L-744,832 caused inhibition of tumor growth in the absence of systemic toxicity. Although FPTase activity was inhibited in tumors from the treated mice, unprocessed Ki-RasB was not detected. These results demonstrate the utility of the MMTV-Ki-rasB transgenic mice for testing potential anticancer agents. Additionally, the data suggest that although the FTI L-744,832 can inhibit tumor growth in this model, Ki-Ras may not be the sole mediator of the biological effects of the FTI.

Peroxisome proliferator-activated receptors gamma and alpha mediate in vivo regulation of uncoupling protein (UCP-1, UCP-2, UCP-3) gene expression.

A role for peroxisome proliferator-activated receptors, PPAR gamma and PPAR alpha, as regulators of energy homeostasis and lipid metabolism, has been suggested. Recently, three distinct uncoupling protein isoforms, UCP-1, UCP-2, and UCP-3, have also been identified and implicated as mediators of thermogenesis. Here, we examined whether in vivo PPAR gamma or PPAR alpha activation regulates the expression of all three UCP isoforms. Rats or lean and db/db mice were treated with PPAR gamma [thiazolidinedione (TZD)] or PPAR alpha (WY-14643) agonists, followed by measurement of messenger RNAs (mRNAs) for UCP-1, UCP-2, and UCP-3 in selected tissues where they are expressed. TZD treatment (AD 5075 at 5 mg/kg x day) of rats (14 days) increased brown adipose tissue (BAT) depot size and induced the expression of each UCP mRNA (3x control levels for UCP-1 and UCP-2, 2.5x control for UCP-3). In contrast, UCP-2 and UCP-3 mRNA levels were not affected in white adipose tissue or skeletal muscle. Chronic (30 days) low-dose (0.3 mg/kg x day) TZD treatment induced UCP-1 mRNA and protein in BAT (2.5x control). In contrast, chronic TZD treatment (30 mg/kg x day) suppressed UCP-1 mRNA (>80%) and protein (50%) expression in BAT. This was associated with further induction of UCP-2 expression (>10-fold) and an increase in the size of lipid vacuoles, a decrease in the number of lipid vacuoles in each adipocyte, and an increase in the size of the adipocytes. TZD treatment of db/db mice (BRL 49653 at 10 mg/kg x day for 10 days) also induced UCP-1 and UCP-3 (but not UCP-2) expression in BAT. PPAR alpha is present in BAT, as well as liver. Treatment of rats or db/db mice with WY-14643 did not affect expression of UCP-1, -2, or -3 in BAT. Hepatic UCP-2 mRNA was increased (4x control level) in db/db and lean mice, although this effect was not observed in rats. Thus, in vivo PPAR gamma activation can induce expression of UCP-1, -2, and -3 in BAT; whereas chronic-intense PPAR gamma activation may cause BAT to assume white adipose tissue-like phenotype with increased UCP-2 levels. PPAR alpha activation in mice is sufficient to induce liver UCP-2 expression.

Mice deficient for the amyloid precursor protein gene.

To understand the in vivo function of the amyloid precursor protein (APP) we generated an APP null mutation in mice by homologous recombination in embryonic stem (ES) cells. We show here that homozygous APP deficient mice were produced at expected frequencies. Neither APP mRNA nor protein could be detected in these animals. Yet the homozygous APP mutant mice are fertile and do not show overt abnormalities at up to 12 weeks of age. Neuroanatomical studies of the brain did not reveal significant differences in the knockout mice as compared to the wild-type controls. These results argue against an essential function of APP in mouse embryonic and early neuronal development.

Effect of inhibitors of factor Xa or platelet adhesion, heparin, and aspirin on platelet deposition in an atherosclerotic rabbit model of angioplasty injury.

Acute thrombotic reocclusion and restenosis after successful coronary angioplasty are limitations of the procedure. Although the restenotic process is not completely understood, acute platelet deposition and thrombosis are considered important initiating mechanisms. The effort to identify pharmacologic agents capable of modifying acute platelet action following mechanical injury requires an animal model mimicking the clinical pathophysiology as closely as possible. We developed a model of angioplasty-induced injury in atherosclerotic rabbit femoral arteries. Acute 111indium-labelled platelet deposition and thrombosis were assessed four hours after balloon-injury in arteries subjected to prior endothelial damage (air desiccation) and cholesterol supplementation (one month). The effects of recombinant tick anticoagulant peptide (rTAP), a blood coagulation factor Xa (fXa) inhibitor and of recombinant leech antiplatelet protein (rLAPP), a platelet adhesion inhibitor, were compared to heparin (HEP) and aspirin (ASA). Recombinant TAP and HEP, but not rLAPP or ASA, successfully prevented thrombus formation and reduced platelet deposition in balloon-injured vessel segments to levels not significantly different from those observed in the contralateral atherosclerotic non-balloon-injured vessels. Therefore, this model, incorporating balloon catheter dilation of arteries exhibiting neointimal growth and atherosclerotic plaque formation, may be useful for evaluation of possible adjunctive therapies during angioplasty.

Anguidine-induced testicular injury in Lewis rats.

Anguidine (diacetoxyscirpenol, DAS) and other trichothecene mycotoxins are potent inhibitors of protein synthesis and injure organs with rapidly dividing cell populations, including the testis. Testicular structure and function were studied in male Lewis rats 1, 3, 7, 30, 60, and 90 days after exposure at age 12 weeks to anguidine at 1.7 mg/kg body weight given by ip injection. The dose was equivalent to 75% of the ip LD50. Anguidine caused a gradual decline in testicular weight beginning 30 days after treatment. Sperm production was also reduced by 30 days, and the frequency of hypocellular seminiferous tubules increased by day 60. There was no evidence of recovery by 90 days. These changes are consistent with injury to proliferating cells early in the maturation sequence. Epididymal sperm reserves were reduced by 3 days after anguidine administration, prior to the reduction in sperm production, suggesting premature release of spermatozoa from the epididymis.

Comparison of motility and membrane integrity to assess rat sperm viability.

Rat sperm motility and membrane integrity were compared as endpoints for viability. Sperm motility was measured by computer-assisted semen analysis (CASA), whereas membrane integrity was assessed by flow cytometric analysis of sperm stained with two nucleic acid stains, SYBR-14 and propidium iodide. The two techniques were compared in experiments that examined sperm viability over time and by analysis of known mixtures of control and freeze/thaw-killed sperm. Results from the two approaches were quantitatively very similar. Sperm from rats treated with dibromoacetic acid (600 or 1200 mg/kg) or alpha-chlorhyrin (100 mg/kg) were also analyzed. Neither technique detected a treatment-related effect with dibromoacetic acid. CASA identified a significant decrease in sperm motility in alpha-chlorhyrin-treated rats, whereas flow cytometric analysis did not find a measureable change in sperm membrane integrity. Because decreases in sperm motility would be expected to directly affect fertility, CASA may be a more robust endpoint for risk assessment in reproductive toxicology studies than flow cytometric analysis of membrane integrity.

Alcohol and cancer.

The cancers consistently associated with ingestion of alcohol, the head and neck cancers, are also associated with tobacco use and arise from epithelia that are in direct contact with both agents. Tobacco smoking-related cancers at sites not directly in contact with alcoholic beverages, that is, lung, bladder, and perhaps pancreas, do not consistently show a relationship to alcohol consumption, although lung and pancreatic tumors are associated in some studies. Liver cancer was thought to be strongly related to alcohol consumption on epidemiological grounds and because of its relationship to cirrhosis. As knowledge of the viral etiology of some cirrhoses has evolved and as methods to detect viruses have developed, the significant association between hepatitis B virus and hepatocellular carcinoma has become clear. Alcohol and hepatitis B virus may interact in the etiology of the disease and have important separate roles as well. There are epidemiologic and experimental data showing that malnutrition (resulting from poor food choice), economic deprivation, or alcoholism contributes to the risk for head, neck, and liver cancers. Colon cancers occur about equally in men and women, are found in well-nourished populations, and are not associated with tobacco smoking. Rectal cancers show a preponderance of cases in men but are frequently found in women as well and are not thought to be associated with smoking or malnutrition. The association between colorectal cancers and alcohol consumption, when it is found, apparently occurs at even relatively low alcohol intakes and is often stronger for consumption of beer than of other beverages. Nutritional and metabolic mechanisms proposed for the influence of alcohol on carcinogenesis are supported by studies in human subjects and laboratory animals. Animal models are needed in which effects of ethanol on carcinogenesis can be consistently demonstrated and which can then be used to examine mechanisms.

Tuberculosis in five basset hounds.

Five Basset Hounds (2 females and 3 males) under the age of 5 years, acquired systemic tuberculosis. We suspected tuberculosis in one dog, because it had histologic lesions similar to those in 4 dogs in which bacteria were identified as Mycobacterium avium complex. A review of canine tuberculosis revealed a similar diagnosis in a Basset Hound. The association of this infection in Basset Hounds suggests an inherited immunologic defect. Results of our survey suggest that the defect might exist in cell-mediated immunity.

Nutrient influences on toxicity and carcinogenicity.

Essential nutrients and toxicants often coexist in the diet of humans and animals. Interactions among nutrients and chemicals in the diet may alter the toxicity of the chemical or the requirement for or availability of specific nutrients. Such interactions have been demonstrated among toxins and dietary protein, trace elements, and vitamins. Certain nutrients may also alter the response to certain drugs, and some drugs, such as oral contraceptives, may alter the availability of some nutrients. The carcinogenicity of some compounds and the subsequent promotion of tumor growth are affected by nutrients such as fat, vitamins, and lipotropes and by such minor dietary constituents as naturally occurring indoles and synthetic preservatives. In this paper we review such interactions as well as the mechanisms responsible for them.

Food additives and contaminants. An update.

Food additives continue to be a source of benefits to the consuming public but there are also perceived risks. Concern for the latter in the last decade has produced a society afflicted with cancer phobia. The intentional additives including sugars, salt, corn syrup, and dextrose make up 90% of the direct additives. These, along with a limited number of familiar items make up a large proportion of the remainder of the additives. Such common ingredients as nitrates and nitrites, solanine, cyanogenetic compounds, arsenic, etc., are unavoidably consumed in the diet and with little if any evidence for public health consequences. Major concern on the part of the public in recent years has been focused on man-made chemicals which are intentionally added to foods to enhance flavors and acceptability, nutrient value, shelf life and increased availability. These include food colors, nonnutritive and low-nutrient sweeteners, (saccharin, cyclamate, aspartame); antioxidants; and nitrites. Contaminants, sometimes incorrectly included in lists of food additives, present the greatest potential threat to public health. Such contaminants as mycotoxins, nitrosamines, polychlorinated biphenyls (PCBs), pesticides, among others, provide a continuing challenge to our regulatory agencies and to public health authorities. Evidence to date indicate that these responsible for food safety are doing an admirable job, and as a society, our food supply has never been better, or safer, and, as a population, we have never been healthier. Aside from contaminants, major concerns relate to an excess of good food and to obesity. These comments should not be taken to infer that we should relax our concern and surveillance; instead more concern and surveillance should be exerted toward those uncontrolled substances such as natural plant products and alleged natural nutrients, roots, herbs, etc., which are given much credit for positive health effects, without meeting the high standards of our legitimate food industry.

Dietary modifiers of cancer.

The foregoing review of current risk factors associated with single nutrients and nutritional status in human populations, supported by animal studies, point clearly to a possibility for manipulation of nutrients in our diet, which may be a major tool for prevention of cancer. Migrant populations exhibit changes in incidence and frequency of certain types of cancer indicating external causes for cancer with diets high on the list of suspects. The foods we eat are probably the most important risk factors that human populations encounter and they offer a major focus for hope of preventing cancer. Food is also the most complex mixture to which we are exposed. Of all lifestyle factors associated with cancer, diet is one over which we exercise control. Epidemiologic observations, supported by controlled animal model research on carcinogenesis strongly suggest that through dietary control, some types of cancer are preventable. The evidence to date suggest that of all dietary factors of significance, those most likely to affect risk include total calories, protein, fat, vitamin A and carotene, the lipotropic factors methionine and choline, and the minerals zinc and selenium.

Mapping of beta-adrenergic receptors in rat lung: effect of isoproterenol.

beta-Adrenergic receptors were mapped in the rat lung using a light microscopic technique that identifies binding sites for 3H-labeled dihydroalprenolol in intact slide-mounted tissue sections, which were also used for biochemical analysis of ligand binding. Grain density measurements were combined with morphometric data. These results indicate that, although over 97% of the specific binding is to cells of alveolar walls, beta-adrenergic receptors are present and can be quantified in tissues that represent a small fraction of total lung mass, such as bronchial epithelium and smooth muscle of bronchial walls and pulmonary vessels. Repeated administration of isoproterenol decreased the receptor number, as determined biochemically and from grain density measurements, in all anatomic regions studied, but did not alter the distribution of binding.

Drug-nutrient interactions and their implications for safety evaluations.

In order to assess the relevance of the drug-nutrient interactions described in this chapter to routine toxicologic studies, the range of nutrient concentrations within which these interactions may occur must be compared to the range of nutrient concentrations found in routinely used rodent diets. While obviously deficient levels of some nutrients were supplied to demonstrate some of the interactions, others occur when the nutrients are present in adequate or excess levels, such as might be found in commercially available diets. These diets are known to vary from batch to batch in nutrient content. A lifetime toxicity/carcinogenicity bioassay using rodents may last longer than 2 years, during which time several batches of diet will be used. The variation in diet composition, coupled with inadequate diet description, makes nutrient-toxin interactions not only possible, but difficult to recognize. These considerations raise the practical and philosophical question as to what type of diet is most appropriate for rodents used for safety evaluation of drugs and chemicals. Is it appropriate to use diets that vary unpredictably in nutrient content, that infer a degree of protection against chemical carcinogenesis and which supply some nutrients such as protein in great excess of dietary needs? Is the increase in sensitivity to chemical carcinogens of animals fed purified diets desirable or does this increased sensitivity of the bioassay exceed that required to assess the risk of human exposure? In other words, is the use of purified diets likely to increase the number of false positive results? Proper interpretation and extrapolation of safety evaluation studies requires adequate description of the test system. Given the profound influence of diet on the response to some toxins, the composition of the diet should, ideally, be defined with the same rigor as are the test compound and the strain, age, sex, and housing conditions of the animals. It is likely, however, that natural ingredients diets will continue to be the diets of choice in safety evaluation studies. This is largely due to economic reasons. It is possible, however, to use these diets with greater confidence if open-formula diets are used and the concentration of each nutrient is reported. Consideration should also be given to preparing diets for use in adult and aging rodents, diets in which protein content is reduced.(ABSTRACT TRUNCATED AT 400 WORDS)

The influence of food additives and related materials on lower bowel structure and function.

Food additives, drugs, and other chemicals are known to influence the lower gastrointestinal tract under some defined conditions, resulting in morphological alterations in the mucosa and other tissues, deranged absorption and excretion of nutrients, and, in some cases, injury to other organs and tissues as a secondary phenomenon. Generally, in rats, hamsters, and dogs, there is increased absorption and urinary excretion of calcium, soft stools or diarrhea, and cecal enlargement. In the rat, hamster, and dog, renal lesions accompany the hypercalcemia and elevated excretion of calcium. These signs, symptoms, and lesions are typical of exposure to sugar alcohols (sorbitol, mannitol, xylitol, lactitol), lactose and caramel, some of the chemically modified food starches, and synthetic polydextrose. Soft stools and diarrhea, as well as cecal enlargement and variable hyperplasia of the colon mucosa, occur frequently when substances are absorbed incompletely in the small intestine and subjected to microbial metabolism in the cecum and colon. The remarkable cecal enlargement, mucosal hyperplasia and, when present, colonic mucosal hyperplasia are reversible, even when long-standing. Renal lesions are reversible only if exposure is of short duration, before significant mineralization and scarring has occurred. Morphological and functional anomalies associated with some of these substances are described and illustrated.

Lung injury in guinea pigs caused by multiple exposures to ultrafine zinc oxide: changes in pulmonary lavage fluid.

Metal oxide particles with diameters of less than 0.1 micron (ultrafine particles) are important products of fossil fuel combustion. Pulmonary lavage fluid was obtained from guinea pigs given 1, 2, or 3 consecutive, daily, 3-h, nose-only exposures to 0, 2.3, 5.9, or 12.1 mg/m3 of freshly generated zinc oxide (ZnO) particles with a projected area diameter of 0.05 micron. Exposure to ZnO at 5.9 or 12.1 mg/m3 was associated with increased protein, neutrophils, and activities of angiotensin-converting enzyme, alkaline phosphatase, acid phosphatase and lactate dehydrogenase in lavage fluid, and with histologic evidence of pulmonary damage characterized by centriacinar inflammation. The severity of inflammation, graded by the number of inflammatory foci per square centimeter of lung, correlated with the amount of protein and the activity of angiotensin-converting enzyme and other enzymes in lavage fluid. These results indicate that analysis of pulmonary lavage fluid is a useful and sensitive method for quantitative evaluation of pulmonary damage caused by inhalation of low levels of ultrafine ZnO.

Amelioration of bleomycin-induced pulmonary injury by cyclosporin A.

This study evaluated the effect of cyclosporin-A (CyA), a potent immunosuppressive drug, on Bleomycin (Bleo)-induced pulmonary inflammation in hamsters. Pulmonary injury was induced by a single intratracheal (i.t.) instillation of Bleo. Four groups of 10 male Syrian hamsters each received one of four treatments: (1) i.t. Bleo and daily intraperitoneal (i.p.) injections of CyA starting 1 day before i.t. instillation of Bleo (Bleo-CyA); (2) i.t. Bleo and i.p. injections of saline (Bleo-Sal); (3) i.t. saline and i.p. CyA (Sal-CyA); (4) i.t. saline and i.p. saline (Sal-Sal). Animals were sacrificed 14 days after i.t. treatment. Lung injury was evaluated histologically, biochemically, and by analysis of bronchoalveolar lavage (BAL) fluid. Treatment of hamsters with CyA significantly ameliorated the Bleo-induced lung injury, as determined by a semiquantitative morphological index that assesses the severity and extent of the injury on a scale of 0-3. Lung hydroxyproline measurements were lower in Bleo-CyA compared to Bleo-Sal, comparable to Sal-Sal and Sal-CyA controls. The percentage of neutrophils, eosinophils, and lymphocytes in BAL fluid was higher in Bleo-Sal and Bleo-CyA animals when compared with control Sal-CyA or Sal-Sal animals. A further increase in percentage of eosinophils was observed in Bleo-CyA compared with Bleo-Sal animals (13.3 +/- 6.6% [mean +/- SE] and 3.7 +/- 2.1%, respectively, p = .0007). BAL fluid protein content was higher in Bleo-Sal compared to Sal-Sal animals, but BAL fluid protein content from Bleo-CyA was not significantly different from that of Bleo-Sal animals. These results indicate that CyA ameliorates the Bleo-induced inflammation but does not prevent leakage of plasma protein or cells into the airspaces. The increased eosinophil numbers in Bleo-CyA-treated hamsters suggests enhanced production of interleukin-4 and -5.