NMR-based metabolic profiling of urine, serum, fecal, and pancreatic tissue samples from the Ptf1a-Cre; LSL-KrasG12D transgenic mouse model of pancreatic cancer.

Pancreatic cancer is the third leading cause of cancer deaths in the United States with more than 53,000 expected to be diagnosed with the disease in 2018. The median survival time after diagnosis is four to six months. The poor survival statistics are due in part to the fact that pancreatic cancer is typically asymptomatic until it reaches advanced stages of the disease. Although surgical resection provides the best chance of survival, pancreatic cancer is rarely detected when surgery is still possible due, in part, to lack of effective biomarkers for early detection. The goal of the research reported here was to determine if it was possible to identify metabolic biomarkers for detection of pre-cancerous pancreatic intraepithelial neoplasia (PanIN) that precede pancreatic adenocarcinoma. The transgenic Ptf1a-Cre; LSL-KrasG12D mouse strain was used as a model of pancreatic cancer progression. Nuclear magnetic resonance (NMR) spectroscopy was employed to compare metabolic profiles of urine, sera, fecal extracts, and pancreatic tissue extracts collected from control and study mice aged 5, 11, and 15 months, including 47 mice with tumors. We were able to identify the following potential biomarkers: decreased 3-indoxylsulfate, benzoate and citrate in urine, decreased glucose, choline, and lactate in blood, and decreased phenylalanine and benzoate and increased acetoin in fecal extracts. Potential biomarkers were validated by p-values, PLS-DA VIP scores, and accuracies based on area under ROC curve analyses. Essentially, all of the metabolic profiling changes could be explained as being associated with the consequences of bicarbonate wasting caused by a complete substitution of the normal pancreatic acinar tissue by tissue entirely composed of PanIN. Given the nature of the mouse model used here, our results indicate that it may be possible to use NMR-based metabolic profiling to identify biomarkers for detection of precancerous PanIN that immediately precede pancreatic cancer.

Infection with Opisthorchis felineus induces intraepithelial neoplasia of the biliary tract in a rodent model.

The liver fluke Opisthorchis felineus is a member of the triad of epidemiologically relevant species of the trematode family Opisthorchiidae, and the causative agent of opisthorchiasis felinea over an extensive range that spans regions of Eurasia. The International Agency for Research on Cancer classifies the infection with the liver flukes Opisthorchis viverrini and Clonorchis sinensis as group 1 agents and a major risk factor for cholangiocarcinoma. However, the carcinogenic potential of the infection with O. felineus is less clear. Here, we present findings that support the inclusion of O. felineus in the Group 1 list of biological carcinogens. Two discrete lines of evidence support the notion that infection with this liver fluke is carcinogenic. First, novel oxysterol-like metabolites detected by liquid chromatography-mass spectroscopy in the egg and adult developmental stages of O. felineus, and in bile, sera, and urine of liver fluke-infected hamsters exhibited marked similarity to oxysterol-like molecules known from O. viverrini. Numerous oxysterols and related DNA-adducts detected in the liver fluke eggs and in bile from infected hamsters suggested that infection-associated oxysterols induced chromosomal lesions in host cells. Second, histological analysis of liver sections from hamsters infected with O. felineus confirmed portal area enlargement, inflammation with severe periductal fibrosis and changes in the epithelium of the biliary tract characterized as biliary intraepithelial neoplasia, BilIN. The consonance of these biochemical and histopathological changes revealed that O. felineus infection in this rodent model induced precancerous lesions conducive to malignancy.

Effect of zinc and copper supplementation on the prognostic value of urinary 5-methyl-2'- deoxycytidine in DMBA-induced carcinogenesis in rats.

BACKGROUND: Epigenetic alterations have been identified as promising new targets for cancer prevention strategies as they occur early during carcinogenesis and represent potentially initiating events for cancer development. OBJECTIVE: The aim of the present study was to assess the effect of zinc and copper on the DNA methylation in rats whose breast adenocarcinoma was simultaneously induced with 7, 12 dimethylbenz[a]anthracene (DMBA). The research focused on the kinetics of alterations in urinary 5-MedC (5-methyl-2'-deoxycytidine) at the early and late stages of carcinogenesis, as well as the influence of dietary factors on the process. METHODS: The content of 5-methyl-2'-deoxycytidine in the rats' urine was determined by the ELISA (enzyme-linked immunosorbent assay) method. The 5-MedC level was standardized by conversion to the creatinine level. RESULTS: It was found that in the rats fed only the standard diet and DMBA-treated the urinary levels of 5-MedC collected after the 10th week were considerably lower in comparison with the content of this biomarker in urine starting from the 19th week (43.56 ± 14.34 vs. 71.84 ± 42.64). The animals treated with DMBA and additionally obtaining copper were characterized by a much higher content of the examined biomarker in urine, both in the early phase of carcinogenesis (10th week) and later (19th week), as compared with the animals fed only the standard diet or the zinc-supplemented diet. In the rats with a fully developed tumor (100% incidence of the disease) the applied dose of resveratrol (0.2 mg/kg bw) was too low to prevent the intensive formation and increase of 5-MedC level in urine, additionally stimulated by the presence of Cu in the diet as well as by the active, ongoing neoplastic process. CONCLUSIONS: Summing up the obtained results of investigations it can be said that the urinary level of 5-MedC depends on the applied supplementation.

Comparative pharmacokinetics of RAD001 (everolimus) in normal and tumor-bearing rodents.

PURPOSE: Comparative pharmacokinetic (PK) analysis of the mTOR inhibitor RAD001 (everolimus) in rats and mice. METHODS: Blood cell partitioning, plasma protein binding and PK parameters of RAD001 in blood and tissues (including brain) of both mice and rats were determined. PK modeling predicted plasma/blood and tumor levels from a variety of regimens and these were compared with the known human PK profile. DCE-MRI was used to compare tumor vascularity between mice and rats. Estimation of IC50 values in vitro and ED50 values in vivo were used to provide an indication of anti-tumor activity. RESULTS: The PK properties of RAD001 differed between mice and rats, including erythrocyte partitioning, plasma protein binding, plasma/blood t(1/2), oral bioavailability, volume of distribution, tissue/tumor penetration and elimination. Modeling of tumor and blood/plasma PK suggested that in mice, multiple daily administrations result in a 2-fold increase in tumor levels of RAD001 at steady state, whereas in rats, a 7.9-fold increase would occur. Weekly high-dose regimens were predicted not to facilitate tumor accumulation in either species. Total tumor levels of RAD001 were four- to eight-fold greater in rats than in mice. Rat tumors had a >2-fold greater plasma content and permeability compared to mouse tumors, which could contribute to differences in tumor drug uptake. Maximal antitumor effects (T/C of 0.04-0.35) were observed in both species after daily administration with similar C(max) and AUC values of unbound (free) RAD001. These free levels of RAD001 are exceeded in serum from cancer patients receiving clinically beneficial daily regimens. In rodents, brain penetration of RAD001 was poor, but was dose-dependent and showed over-proportional uptake in rats with a longer t(1/2) compared to the systemic circulation. CONCLUSIONS: The PK of RAD001 differed between mice and rats, with rats having a PK profile closer to that of humans. High intermittent doses of RAD001 may be more appropriate for treatment of brain tumors.

A mouse model repository for cancer biomarker discovery.

Early detection of cancer using biomarkers obtained from blood or other easily accessible tissues would have a significant impact on reducing cancer mortality. However, identifying new blood-based biomarkers has been hindered by the dynamic complexity of the human plasma proteome, confounded by genetic and environmental variability, and the scarcity of high quality controlled samples. In this report, we discuss a new paradigm for biomarker discovery through the use of mouse models. Inbred mouse models of cancer recapitulate many critical features of human cancer, while eliminating sources of environmental and genetic variability. The ability to collect samples from highly matched cases and controls under identical conditions further reduces variability which is critical for successful biomarker discovery. We describe the establishment of a repository containing tumor, plasma, urine, and other tissues from 10 different mouse models of human cancer, including two breast, two lung, two prostate, two gastrointestinal, one ovarian, and one skin tumor model. We present the overall design of this resource and its potential use by the research community for biomarker discovery.

Seasonal rhythms of 6-sulphatoxymelatonin (aMT6s) excretion in female rats are abolished by growth of malignant tumors.

The effect of development and growth of malignant tumors on pineal melatonin production was studied in two different hormone-dependent tumor systems in female rats. Urinary excretion of 6-sulphatoxymelatonin (aMT6s), the metabolic end product of melatonin, which parallels its production, was determined by radioimmunoassay at fortnightly or monthly intervals over the period of 1 year in female F344 Fischer rats bearing chemically-induced mammary carcinomas and in BDII/Han rats with spontaneous endometrial carcinomas. Untreated Fischer rats and BDII/Han rats in which tumor growth was suppressed by treatment with a progestin served as controls. Based on the cosinor analysis, animals without tumors showed significant seasonal rhythms of aMT6s excretion, with peaks in August (Fischer rats) and in May (BDII/Han rats). In contrast, such rhythms were absent in animals with developing and manifest tumors. It is concluded that animals kept under constant environmental conditions still show seasonal rhythms of pineal activity. Tumor development and growth affect pineal activity leading to disturbance of these rhythms.

ESR imaging on a solid-tumor-bearing mouse using spin-labeled dextran.

Imaging of a tumor with ESR was tried using two different types of spin probes, a low molecular weight spin probe, CPROXYL, and a polymer spin probe, TEMPO-DX. Spin probes were administered to a mouse bearing a solid tumor that was a transplanted Ehrlich's ascites carcinoma in the back, using two methods, conventional intraperitoneal injection and continuous intravenous injection with a micro-feeder. First, the accumulation of the probe was examined by X-band ESR. CPROXYL, which was administered to a mouse intraperitoneally, was exclusively retained in urine, showing that it was rapidly excreted into the bladder, while TEMPO-DX was absorbed from the peritoneal cavity with difficulty to the vessel. Using continuous intravenous injection, CPROXYL was also rapidly excreted, but it was confirmed that TEMPO-DX concentrated in tumor tissue because it has a long half-life in vivo. In addition, measurement of ESR imaging was done to measure the distribution of spin probes with continuous intravenous injection. The strongest spot of CPROXYL was observed on ESR images, showing the accumulation at the bladder, while the spot of TEMPO-DX was observed in the solid tumor of the back of the mouse. These results suggest that TEMPO-DX could stay much longer than a low molecular weight spin probe in vivo and concentrate at the tumor. TEMPO-DX may be useful for developing specific ESR imaging agents for tumor.

Analysis of potential biomarkers of estrogen-initiated cancer in the urine of Syrian golden hamsters treated with 4-hydroxyestradiol.

Estrone (E1) and 17beta-estradiol (E2) are metabolized to catechol estrogens (CE), which may be oxidized to semiquinones and quinones (CE-Q). CE-Q can react with glutathione (GSH) and DNA, or be reduced to CE. In particular, CE-3,4-Q react with DNA to form depurinating adducts (N7Gua and N3Ade), which are cleaved from DNA to leave behind apurinic sites. We report the determination of 22 estrogen metabolites, conjugates and adducts in the urine of male Syrian golden hamsters treated with 4-hydroxyestradiol (4-OHE2). After initial purification, urine samples were analyzed by HPLC with multichannel electrochemical detection and by capillary HPLC/tandem mass spectrometry. 4-Hydroxyestrogen-2-cysteine [4-OHE1(E2)-2-Cys] and N-acetylcysteine [4-OHE1(E2)-2-NAcCys] conjugates, as well as the methoxy CE, were identified and quantified by HPLC, whereas the 4-OHE1(E2)-1-N7Gua depurinating adducts and 4-OHE1(E2)-2-SG conjugates could only be identified by the mass spectrometry method. Most of the administered 4-OHE2 was metabolically converted to 4-OHE1. Formation of thioether (GSH, Cys and NAcCys) conjugates and depurinating adducts [4-OHE1(E2)-1-N7Gua] indicates that oxidation of 4-CE to CE-3,4-Q and subsequent reaction with GSH and DNA, respectively, do occur. The major conjugates in the urine were 4-OHE1(E2)-2-NACCYS: The oxidative pathway of 4-OHE1(E2) accounted for approximately twice the level of products compared with those from the methylation pathway. The metabolites and methoxy CE were excreted predominantly (>90%) as glucuronides, whereas the thioether conjugates were not further conjugated. These results provide strong evidence that exposure to 4-OHE1(E2) leads to the formation of E1(E2)-3,4-Q and, subsequently, depurinating DNA adducts. This process is a putative tumor initiating event. The estrogen metabolites, conjugates and adducts can be used as biomarkers for detecting enzymatic oxidation of estrogens to reactive electrophilic metabolites and possible susceptibility to estrogen-induced cancer.

The excretion of biotrace elements using the multitracer technique in tumour-bearing mice.

A radioactive multitracer solution obtained from the nuclear reaction of selenium with 25 MeV/nucleon 40Ar ions was used for investigation of trace element excretion into the faeces and urine of cancerous mice. The excretion rates of 22 elements (Na, K, Rb, Mg, Ca, Sr, Ga, As, Sc, V, Cr, Mn, Co, Fe, Y, Zr, Mo, Nb, Tc, Ru, Ag and In) were simultaneously measured under strictly identical experimental conditions, in order to clarify the excretion behavior of these elements in cancerous mice. The faecal and urinary excretion rates of Mg, Sr, Ga, As, Sc, V, Cr, Mn, Co, Fe, Y, Zr, Nb, Ru and Mo in cancerous mice, showed the in highest value at 0-8 hours. The accumulative excretion of Ca, Mo, Y and Zr was decreased and Na, Fe, Mn and Co increased in tumour-bearing mice, when compared to normal mice.

Melatonin in cancer patients and in tumor-bearing animals.

A review of findings is given which relate to the levels of circulating melatonin as well as the urinary excretion of its main peripheral metabolite 6-sulphatoxymelatonin (aMT6s) in patients with different types of cancer as well as in tumor-bearing animals. Clinical results show that circulating melatonin tends to be depressed in patients with primary tumors of different histological types including both endocrine-dependent (mammary, endometrial, prostate cancer) and endocrine-independent tumors (lung, gastric, colorectal cancer). Reduction of melatonin is most pronounced in patients with advanced localized primary tumors, such as mammary and prostate cancer where a clear negative correlation with tumor-size exists. The phenomenon of a reduction of circulating melatonin appears to be a transient one since patients with recidives show a normalization of melatonin. Surgical removal of the primary tumor does, however, not lead to normalization indicating that complex systemic changes appear to be involved in the down-regulation of melatonin. It is unclear at present, whether circulating melatonin is depleted in cancer patients due to a reduced production by the pineal gland or due to certain peripheral metabolic processes, although no evidence for an enhanced hepatic degradation to aMT6s, the main peripheral metabolite of melatonin, was found. The reduction of circulating melatonin is accompanied by neuroendocrine changes affecting the circadian secretion of the adenohypophyseal hormones prolactin, somatotropin and thyroid-stimulating hormone. In contrast to the above-described types of tumors many patients with ovarian cancer show highly elevated levels of melatonin perhaps due to the production of tissue-specific growth factors that could affect pineal melatonin secretion. Experiments with tumor-bearing animals clearly demonstrate that nocturnal circulating melatonin is modulated due to malignant growth. Detailed investigations with chemically induced mammary tumors in rats and serial transplants derived thereof show that slow-growing and well-differentiated tumors containing epithelial cell elements (adenocarcinomas and carcinosarcomas) lead to an enhanced production of melatonin involving activation of the rate-limiting enzyme of pineal melatonin biosynthesis (serotonin N-acetyltransferase) probably due to elevation of the sympathetic tone in response to a stimulation of the cellular immune system by malignant growth. As opposed to that nocturnal melatonin is depleted in animals with fast-growing mammary tumor transplants when myoepithelial-mesenchymal conversion leads to pure sarcomas. The reduction of melatonin appears to be due to either a reduced availability of the precursor amino acid tryptophan because of a glucocorticoid-induced activation of the hepatic enzyme tryptophan 2,3-dioxygenase or a direct peripheral degradation of melatonin via indoleamine 2,3-dioxygenase expressed in tumor and/or other tissues. The significance of these clinical and experimental findings relating to melatonin is discussed both in terms of their practical application as a possible tumor marker and from a theoretical point of view to understand better the mechanisms involved in complex host-tumor interactions involving the neuroimmunoendocrine network.

Purification and characterization of a tumor lipid-mobilizing factor.

Cancer patients with weight loss showed urinary excretion of a lipid-mobilizing factor (LMF), determined by the ability to stimulate lipolysis in isolated murine epididymal adipocytes. Such bioactivity was not detectable in the urine of cancer patients without weight loss or in normal subjects. The LMF was purified using a combination of ion exchange, exclusion, and hydrophobic interaction chromatographies to give a single component of apparent Mr 43,000, which showed homology in amino acid sequence with human plasma Zn-alpha2-glycoprotein. Both substances showed the same mobility on denaturing and nondenaturing gels and the same chymotrypsin digestion pattern, both stained heavily for carbohydrate, and they showed similar immunoreactivity. Polyclonal antisera to human plasma Zn-alpha2-glycoprotein was also capable of neutralization of the bioactivity of human LMF in vitro. Using competitive PCR to quantify expression of Zn-alpha2-glycoprotein, we found that only those tumors that were capable of producing a decrease in carcass lipid expressed mRNA for Zn-alpha2-glycoprotein. These results provide strong evidence to suggest that tumor production of Zn-alpha2-glycoprotein is responsible for the lipid catabolism seen in cancer patients.

Individual values of excretion of benzo[a]pyrene metabolites and susceptibility to its carcinogenic effect in rats.

In white outbred LIO rats exposed to multiple intraperitoneal (i.p.) doses (10 mg/kg) of benzo[a]pyrene (BP) in the form of a water-lipid emulsion, individual peculiarities of the excretion of its metabolites, BP-7,8-diol and 3-hydroxy-BP (3-OH-BP) in urine and feces were detected and compared with the carcinogenic effect. Parameters of BP metabolite excretion differed from those found in our previous experiments with rats exposed to single high i.p. doses of BP (100 and 200 mg/kg), dissolved in sunflower oil [11,12]. In comparison with our previous observation, in the present study, the carcinogenic effect was considerably weaker (5/22 versus 10/19). The rats that developed tumours of internal tissues (four peritoneal malignant histiocytomas and one lung lymphosarcoma), excreted higher quantities of BP-7,8-diol in the urine than other rats. The possible implication of monitoring excretion of BP metabolites for predicting individual susceptibility to its carcinogenic effect is discussed.

Basic fibroblast growth factor secreted by an animal tumor is detectable in urine.

Basic fibroblast growth factor (bFGF or FGF-2) is abnormally elevated in the serum and urine of patients with many types of cancer. However, the source of the bFGF is unclear. We developed a model that could distinguish between tumor-derived and host-derived bFGF. We gave athymic mice s.c. injections of cells of the murine K1000 tumor, which secretes a bFGF mutein (bFGF CS23) as its dominant angiogenic factor. Controls were given injections of Lewis lung carcinoma or saline. Urine was collected for 9 weeks, and bFGF was quantitated using two immunoassays which can discriminate between tumor bFGF CS23 and native bFGF. None of the mice had detectable urinary native bFGF, and no control mice had detectable urinary bFGF CS23. In contrast, urine from mice bearing the K1000 tumor revealed detectable bFGF CS23 by 2 weeks, and bFGF CS23 increased with increasing tumor volume throughout the study. Because bFGF CS23 is not produced by other cells, the bFGF CS23 in the urine most likely came from the K1000 tumor and not from the host. These results suggest that the source of elevated bFGF in the urine of human cancer patients is, at least in part, the tumor itself.

Drug metabolism in rats with cancer induced by N-nitrosodiethylamine and phenobarbital.

The metabolism of R- and S-warfarin in vivo and in vitro, bufuralol in vitro, and antipyrine and debrisoquine in vivo were studied in rats with cancer induced by N-nitrosodiethylamine and phenobarbital treatment. Microsomal cytochrome P-450 content was greatly reduced in both healthy and cancerous parts of the livers of tumour-bearing animals. The specific activities of R-warfarin and bufuralol 1'-hydroxylases were significantly elevated in rats with cancer. The activities of S-warfarin hydroxylases expressed per mg microsomal protein were reduced in animals with cancer, whereas those of R-warfarin and bufuralol 1'-hydroxylases were not. The urinary excretion of R-7-hydroxywarfarin was increased and those of S-6- and S-4'-hydroxywarfarin decreased in rats with cancer. The correlations between microsomal formation and urinary excretion of all warfarin metabolites were poor, except for R-7-hydroxywarfarin. Antipyrine oxidation was increased in the cancerous state but the urinary metabolic profiles were similar in rats with cancer and in controls. The metabolism of debrisoquine was decreased in tumour-bearing animals. Antipyrine metabolism did not show any correlation with either warfarin or debrisoquine metabolism, whereas several relationships were observed between warfarin and debrisoquine metabolism and between warfarin and bufuralol metabolism.

Urinary excretion of parathyroid hormone-related protein fragments in patients with humoral hypercalcemia of malignancy and hypercalcemic tumor-bearing nude mice.

To investigate whether parathyroid hormone-related protein (PTHrP), a hypercalcemia-inducing factor responsible for malignancy-associated hypercalcemia (MAH), is excreted into urine of these patients, radioimmunoassay was established using antiserum specific for the C-terminal region of PTHrP-(127-141). Immunoreactive PTHrP (iPTHrP) was detected in the urine of all patients with MAH (n = 6) in whom nephrogenous cyclic AMP excretion was elevated. However, iPTHrP was not detected in the urine of normal subjects (n = 25) or hypercalcemic patients with primary hyperparathyroidism (n = 8). In normocalcemic patients with malignant disorders iPTHrP was not detected in the urine in most cases (24 of 25 patients) but was detectable in 1 of 25 patients. iPTHrP was also detected in the urine of hypercalcemic nude mice transplanted with PTHrP-producing tumors, but not in the urine of control and normocalcemic nude mice transplanted with PTHrP-nonproducing tumor. Furthermore, size-exclusion high-performance liquid chromatography revealed that the molecular weight of iPTHrP is about 2000-6000 daltons in the urine of patients as well as tumor-bearing nude mice. These data indicate that the fragments of the C-terminal region of PTHrP are excreted into the urine of patients with MAH and in a few normocalcemic patients with malignancies, suggesting that the measurement of iPTHrP in the urine is potentially useful in the differential diagnosis of hypercalcemia, particularly in differentiating humoral hypercalcemia of malignancy and primary hyperparathyroidism.

Increased biopterin production in rats with tumors induced by radon inhalation and benzonaphthoflavone administration.

Serial determinations of urinary biopterin were performed in rats during the development of lung tumors induced by radon inhalation and 5,6-benzonaphtoflavone administration. A striking increase in biopterin levels was observed in animals which developed single or multiple epidermoid carcinoma of the lung and this increase occurred several weeks before tumors could be detected radiographically.

Inhibitory action of alpha-difluoromethylornithine on N-butyl-N-(4-hydroxybutyl)nitrosamine-induced rat urinary bladder carcinogenesis.

We previously have shown that urine components capable of stimulating ornithine decarboxylase activity of urothelium can enhance rat urinary bladder carcinogenesis, and that alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase, suppresses carcinogen-initiated rat urinary bladder carcinogenesis. The present investigation was conducted to determine whether DFMO's suppressive effect is stage specific during carcinogenesis and whether the suppressive effect lasts with its continued use. Following initiation with 0.05% N-butyl-N-(4-hydroxybutyl)-nitrosamine in drinking water for 6 wk, male Fischer 344 rats initially weighing 125 to 150 g were randomly divided into two groups, the first receiving 0.2% DFMO in drinking water ad libitum and the second receiving tap water only. Groups of animals were killed at regular intervals until the completion of the experiment at 75 wk. The effect of DFMO was evaluated by monitoring the incidence of tumors, the mean number of tumors per rat, the mean volume of individual tumors, and the mean total tumor volume per rat. The results showed that continuous treatment with DFMO significantly reduced tumor formation until 60 wk (P less than 0.017). The effect was only of borderline significance (0.017 less than P less than 0.035) at 75 wk. Discontinuation of DFMO treatment at 40 wk resulted in the loss of protective effect in all comparisons except for the borderline effect on the tumor number and total tumor volume per rat. DFMO had no significant effect on the incidence or development of preneoplastic early lesions. Mucosal polyamine (spermidine and spermine) levels were reduced and correlated well with the reduction in tumor growth, suggesting that the reduction in tumor growth rate by DFMO may be due to its ability to reduce polyamine levels in urothelium. There were no side effects attributable to DFMO treatment. DFMO may be a useful chemopreventive agent to retard the recurrence of human superficial bladder cancer.

Variant forms of rat epidermal growth factor present in the urine of nude rats bearing human tumors.

Epidermal growth factor (EGF) receptor-binding peptides from the urine of tumor patients have been reported to differ in molecular weight and relative hydrophobicity from those of normal individuals. Nude rats bearing human large cell lung carcinomas or chondrosarcomas and non-tumor-bearing sibling control rats were used to investigate the contributions of tumor and host to urinary EGF-related peptide growth factors. Peptides were adsorbed from urine onto methyl-bonded silica and eluted according to their relative hydrophobicity by a stepwise gradient of aqueous acetonitrile. Total EGF receptor-binding activity relative to urinary creatine was elevated in the urine of only one group of tumor-bearing rats. However, the proportion of relatively hydrophilic activity was increased markedly in all three groups of tumor-bearing rats. Rats bearing a large cell lung cancer excreted unusually hydrophilic Mr 6000 peptides that were chromatographically similar to transforming growth factor alpha on reverse phase high performance liquid chromatography but proved to be rat EGF by radioimmunoassay (RIA). EGF receptor-binding activity that was common to the urine of tumor-bearing animals regardless of tumor type, but more hydrophilic than that from control rats, had Mr 60,000, 30,000, 12,000, and 4,000 to 7,000 components. All reacted fully in the rat EGF RIA and were negative for human EGF and transforming growth factor alpha by RIA. A more hydrophobic fraction of EGF receptor-binding activity, common to control and tumor-bearing animals, contained Mr 33,000, 5,000 to 7,000, and 2,000 to 5,000 components. High performance liquid chromatography and gel electrophoresis of the Mr 33,000 activity revealed a high molecular weight rat EGF comparable to that reported in human urine. No human EGF or transforming growth factor alpha was detected by RIA in any of the active fractions from tumor-bearing rat urine. Thus, all EGF receptor-binding activity appeared to derive from rat EGF produced by the rat host and not by the xenografted tumors.

Polyamine metabolism and polyamine excretion in normal and tumor bearing rodents.

Aminoguanidine sulfate (AG) inhibits in vivo oxidative deaminations of the polyamines and their derivatives. This compound was used to study urinary polyamine excretion by normal, and tumor bearing rodents. Of the total expendable polyamines, 64 percent were catabolized by AG-sensitive oxidases and escaped observation. Tumor bearing animals did not excrete enhanced amounts of polyamines at any stage of tumoral growth. However, treatment with adriamycin caused an increased polyamine excretion. Prolonged administration of a 2% solution of a-difluoromethylornithine (DFMO), reduced urinary polyamine excretion to the same level of about 27%, irrespective whether the animals carried a large tumor or not. Cadaverine excretion was not affected by treatment with DFMO. Based on these animal data, it appears that urinary polyamines are of restricted value in the diagnosis of tumors.

Comparison of growth factors functionally related to epidermal growth factor in the urine of normal and human tumor-bearing athymic mice.

Urine from nude mice contains epidermal growth factor (EGF) and a minor acid-stable component with an apparent molecular weight of 20,000, which competes with EGF for binding to EGF membrane receptors and which promotes colony formation by normal rat kidney cells in soft agar. The levels of this Mr 20,000 urine-derived growth factor are increased approximately 4- to 10-fold in nude mice bearing tumors following s.c. injection of cultured human tumor cells. Following removal of the primary tumor, the concentration of this factor is reduced to basal levels, and thus, elevated levels of this growth factor appear to be dependent on tumor burden. The Mr 20,000 urinary component is separable into four EGF competing activities by high-performance liquid chromatography; the major species is immunologically related to mouse submaxillary gland EGF and therefore appears to be of host origin. However, in addition to elevated levels of host growth factor, urine from tumor-bearing mice also contains transforming growth factor activity in amounts comparable to that released by the tumor cells in culture. The tumor-derived urinary transforming growth factor activity is immunologically unrelated to EGF but is immunoreactive with an antiserum to transforming growth factor-alpha. We propose that the nude mouse may be a useful model to examine the role of both host- and tumor-derived growth factors in tumorigenesis and the usefulness of these factors as biological markers of response to therapy and tumor progression.