An experimental model of diabetes and cancer in rats.

The aim of this study was to develop an experimental model for the study of cancer associated with diabetes. For diabetes induction, Sprague-Dawley rats were given streptozotocin (STZ, 90 mg/kg body weight (BW), by intraperitoneal injection on the second day of life. For mammary tumour induction, rats were injected with 50 mg/kg BW of N-nitroso-N-methylurea (NMU) at 50, 80 and 110 days old. The neoplastic process and the effect of tamoxifen treatment was examined in non-diabetic and diabetic rats. The latency period, NMU-induced tumour incidence and the number of tumours per rat in diabetic rats versus controls were 117 +/- 7 days versus 79 +/- 9 days (P < 0.001); 93% versus 95% (NS); and 5.2 +/- 1.6 versus 2.7 +/- 0.5 (P < 0.02). A more benign histological pattern for tumours in diabetic animals was observed. Mammary tumours in diabetic rats grew more slowly than in controls. Tamoxifen (1 mg/kg/day) treated diabetic rats showed tumour regression in 67% of NMU-induced mammary tumours versus 53% in controls (NS). Our results show that tumour progression seems to be affected by diabetes in this experimental model. We suggest this is the result of changes to insulin-like growth factors and their receptors, which occur in diabetics, and our future research will examine this hypothesis.

Receptors characterization of intraperitoneally N-nitroso-N-methylurea-induced mammary tumors in rats.

Mammary adenocarcinomas induces in female Sprague-Dawley rats by three intraperitoneal injections of N-nitroso-N-methylurea were studied in order to characterize their estrogen (ER), progesterone (PgR), prolactin (PRLR) and epidermal growth factor (EGFR) receptors. All samples evaluated showed the presence of ER and PgR in the cytosol fraction and PRLR amd EGFR in the membrane fraction. Q (fmol/mg) and K(d) (nM) values were as follows: ER, 56 +/- 11 and 0.5 +/- 0.1; PgR, 109 +/- 25 and 2.2 +/- 0.5 and PRLR, 335 +/- 75 and 0.5 +/- 0.2, respectively. In all tumors studied, two specific sites were found for EGFR, one with Q(1) = 22 +/- 9 and K(d1) = 0.6 +/- 0.3, and the other with Q(2) = 125 +/- 33 and K(d2) = 2.1 +/- 0.5. Receptor content was found to be independent of tumor histopathological variety. Displacement index (DI) with estradiol and tamoxifen of [I(3)H]E2-ER binding showed great heterogeneity, with values ranging from 0.01 to1.54. No correlation between ER content and DI values was found. Antiestrogenic binding sites were not found in the microsomal fraction of ten mammary tumors examined. Proliferation of this experimental mammary tumor may be regulated by a complex interaction of steroid and polypeptide hormones, as well as growth factors.

Effect of tamoxifen on intraperitoneal N-nitroso-N-methylurea induced tumors.

The effect of tamoxifen (TAM) was evaluated on a mammary tumor model induced in Sprague-Dawley rats by intraperitoneal administration of three N-nitroso-N-methylurea (NMU) doses. Animals received TAM (1 mg/kg per day) from 10 days before the first NMU dose up to 140 days later. Thereafter, treatment was discontinued and the observation period was extended 60 days longer. Mean overall latency period, tumor number per rat and tumor incidence were recorded. Significant differences between treated and control batches were observed in tumor number per rat (1.8 +/- 1.1 versus 5.2 +/- 1.6; P < 0.05) and in tumor incidence (50% versus 100%; P < 0.05), respectively. No significant difference in latency period between both batches was recorded. All lesions induced in the control batch were malignant, whereas only 45% of those induced in TAM-treated animals were malignant and the remaining 55% were preneoplastic. At 60 days after treatment discontinuance, tumor incidence increased to 90% and also tumor number per rat increased to 4.6 +/- 1.5. TAM effect was also evaluated in rats with NMU-induced tumors by treatment with 1 mg/kg per day during 60 days starting when tumors reached a 1.5-cm diameter. Regression to less than 80% of initial size in 49% of the tumors was observed, while in ovariectomized rats, 33% of tumors regressed. Estrogen receptor content, ER (fmol/mg protein) and Kd (nM) in control tumors were: 56 +/- 10 and 0.5 +/- 0.1. In tumors of TAM-treated animals, ER was less than 5 fmol/mg protein. Findings demonstrate that TAM significantly decreased the appearance of tumors induced in rats by i.p. injection of NMU and when TAM treatment was initiated after tumor induction, some tumors failed to respond to hormonal manipulation. Differential tumor growth response after TAM or oophorectomy in each tumor indicates that in the same rat it is possible to distinguish hormone-dependent and hormone-autonomous tumor populations. Hormonal regulation of tumor growth can be under intrinsic control, regardless of the hormonal status of the whole organism.

Induction of mammary tumors in rat by intraperitoneal injection of NMU: histopathology and estral cycle influence.

In order to obtain an experimental model we induced mammary tumors in female Sprague-Dawley rats. The carcinogen N-nitroso-N-methylurea (NMU) was injected intraperitoneally (i.p.) at doses of 50 mg/kg body weight when animals were 50, 80 and 110 days old. Tumor sizes were measured with a caliper and their growth parameters and histopathological properties were tested. For 100 rats, 88.4% of developed lesions were ductal carcinomas, histologically classified as 52.8% cribiform variety, 30.6% solid carcinoma. Metastases in liver, spleen and lung were present. Other primary tumors were detected with low incidence. The influence of the rat estrous cycle during the first exposure to intraperitoneal NMU injection was studied. The latency period in estrus, proestrus and diestrus was 82 +/- 15, 77 +/- 18 and 79 +/- 18 days, respectively. Tumor incidence was significantly higher in estrus (95.2%) than proestrus (71.4%) or diestrus (77.4), (P < 0.01). Mean number or tumors per animal was similar among the three groups (4.4 +/- 3.2, 3.8 +/- 3.6, 3.2 +/- 1.8). The procedure described appears to be the simplest method for inducing experimental mammary tumors in rats.

H1 and H2 histamine receptors in N-nitroso-N-methylurea (NMU)-induced carcinomas with atypical coupling to signal transducers.

Two specific binding sites for histamine were characterized in the cell membrane of N-nitroso-N-methylurea (NMU)-induced tumors. The first one, with higher affinity (Kd = 4 +/- 2 nM), was further identified as an H2 type, while the lower affinity one (35 +/- 10 nM) corresponded to an H1 receptor. Histamine concentrations up to 50 nM, as well as H2 agonists, significantly enhanced the phosphoinositide turnover by acting through higher affinity H2 receptors. On the other hand, histamine at concentrations over 50 nM and H1 agonists produced a 100% increase in cAMP levels in a response specifically blocked by mepyramine. These H1 and H2 histamine receptors that exhibit different linkages to second messenger systems may prove to be a characteristic of cells with a high proliferating capacity, such as undifferentiated or transformed cells.

Epidermal growth factor in NMU-induced mammary tumors in rats.

In this work we analyze the hypothesis that tumors induced by i.p. N-nitroso-N-methylurea injection express EGF-like peptides and EGF receptors which could be involved in the response to hormone manipulation. EGF receptors (EGFR) were determined in the purified membrane fraction of tumors from control and ovariectomized (OVX) animals and no significant differences were found in either maximal binding capacities (Q) or dissociation constants (Kd) between them. Neither did we observe differences between tumors that regressed (HR) or continued growing (HU) after ovariectomy. In order to test the ability of EGFR to trigger a biological response we measured the production of second messengers inositol triphosphates (IP3) and cAMP levels; we found that EGF increases IP3 production in a dose-dependent way, while cAMP levels were not affected. In addition, EGF was able to induce in vitro cell proliferation in a concentration-dependent manner when tested in primary cultures of tumor cells by the clonogenic soft agar technique. EGF/TGF-alpha activity was determined by a radioreceptor assay in tumor cytosols from control and OVX rats. Results showed a trend to lower values in tumors from OVX rats, but no differences between HR and HU tumors. A positive correlation was found between EGF/TGF-alpha activity and progesterone receptor maximal binding capacity. When we tested the action of estradiol and EGF added together to primary cultures of tumor cells we found an additive effect on cell proliferation. The study of steady state mRNA levels showed that E2 increases PgR and c-myc mRNA levels in HR but not in HU tumors. In conclusion, the autocrine loop EGFR-EGF/TGF-alpha present in all tumors is hormonally regulated, possibly by Pg, but is not related to the tumor response to ovariectomy.

Histamine as an autocrine growth factor: an unusual role for a widespread mediator.

The involvement of histamine in cancer growth represents an old controversy and direct experimental evidence proving this hypothesis is not still available. In this paper we review the most relevant mechanisms referring to the role of histamine receptors, histidine decarboxylase and histamine release in the onset of an autocrine loop, that enables histamine to act as an autocrine growth factor. We postulate that this autocrine loop, that has been studied in an experimental mammary carcinoma model induced in rats, may be present in different human neoplasias. Therefore, the better understanding of this novel regulatory pathway that is controlled by histamine may contribute to identifying new therapeutic targets.

Effect of histamine on growth and differentiation of the rat mammary gland.

The presence of H1 and H2 histamine receptors and their associated second messenger systems were studied during the development of the rat mammary gland. In the tissue of the young female, histamine presented a double receptor site as previously described for experimental mammary tumors, namely a high affinity H2 site (Kd = 10 +/- 2 nM, Bmax = 1068 +/- 71 fm/mg prot.), which mediated its effect via the products of phosphoinositide hydrolysis and a low affinity H1 receptor (Kd1 = 5 +/- 2 nM, Bmax = 188 +/- 33 fm/mg prot. and Kd2 = 41 +/- 20 nM, Bmax = 1980 +/- 790 fm/mg prot. when characterized with 3H-mepyramine), coupled to adenylyl cyclase activation. On the other hand, the mammary gland of the adult rat presented these receptors coupled to the classical second messenger systems described for mammalian cells, that is, the H2 receptor produced an increase in intracellular cAMP levels and the H1 receptor increased the phosphoinositide turnover. We conclude that histamine plays a critical role during development and differentiation of the normal rat mammary gland.

Hormone dependence of mammary tumors induced in rats by intraperitoneal NMU injection.

The purpose of this work was to determine the hormone dependence of mammary tumors induced in Sprague-Dawley rats by three intraperitoneal injections of N-nitroso-N-methylurea at 50, 80, and 110 days of age. Two experimental designs were carried out: (a) Ten days before the first NMU injection, 130 rats were divided into 13 batches and randomly assigned to the following treatments: control, ovariectomy (OVX), tamoxifen (TAM), bromocriptine (BROM), haloperidol (HAL), estradiol (E2), progesterone (Pg), OVX + BROM, TAM + BROM, OVX + HAL, TAM + HAL, OVX + TAM, and E2 + BROM. After 150 days of treatment the following growth parameters were determined: latency period (LP), mean tumor number per rat (n/t), and tumor incidence (TI). LP was significantly increased (p < 0.05) only by Pg and TAM + BROM. The n/t was significantly decreased (p < 0.05) by all treatments except HAL. TI was significantly reduced by OVX, TAM, BROM, and their combinations, (b) Rats bearing ip-NMU-induced mammary tumors were divided into 7 batches and assigned to the following treatments: control, OVX, TAM, BROM, HAL, OVX + BROM, and TAM + BROM. Tumor growth was assessed up to 60 days of treatment; only OVX, TAM and their combination with BROM were able to produce tumor regression. These results support the essential role of E2 and prolactin in the promotion stage of carcinogenesis. However, for established tumors, growth becomes more independent from hormone influence, in particular from prolactin deprivation. We conclude that this model seems suitable for studying the mechanisms underlying the evasion of hormonal control of tumor growth.

Atypical association of H1 and H2 histamine receptors with signal transduction pathways during multistage mouse skin carcinogenesis.

OBJECTIVE: In the present work we studied the association of histamine receptors with second messengers during multistage carcinogenesis in Sencar mice skin. METHODS: 96 Sencar female mouse, divided into six groups were used. Tumors appeared only in the 7, 12-dimethylbenz[a]anthracene-initiated and 12-O-tetradecanoylphorbol-13-acetate-promoted group. Control groups received only TPA, or acetone or no treatment at all. Periodically during the promotion period, cAMP and inositol phosphate production were measured after stimulation with H1 or H2 agonists in samples from all groups. RESULTS: In non-treated skin, H1 receptors were coupled to phosphatidylinositol hydrolysis and H2 receptors mediated cAMP production. Conversely, in tumors H2 receptors were associated with phosphatidylinositol hydrolysis and H1 mediated a rise in cAMP levels. The skin among tumors and the skin from all control groups maintained the same coupling as non-treated skin. An increase in mast cell number, with a homogeneous subepithelial distribution and marked phenotypic changes, was also observed in promoted skin. CONCLUSIONS: These findings indicate an atypical association of histamine receptors with second messengers that could be a critical feature for the postulated action of histamine in tumor growth.

Histamine as an autocrine growth factor in experimental mammary carcinomas.

In order to determine the role of endogenous histamine in the regulation of cell growth, the in vitro action of fluoromethyl-histidine (MFMH) was studied in experimental mammary carcinomas induced in rats. Tumor cells were cultured in soft agar using the clonogenic agar technique. The MFMH was added in different concentrations (0.01-100 microM). The effect observed was a 60% inhibition on colony formation with a maximal effect at concentrations over 10 microM. This action was completely reverted by the H2 agonists dimaprit and arpromidine with an IC50 value of 1 microM. The action of the H2 agonists when added alone was a significant increase in cell proliferation (135%), while the H1 agonist produced a dose-dependent inhibition on cell growth. In these experimental carcinomas endogenous histamine is critical for cell proliferation and one of its major effects may be the stimulation of cell growth by acting on specific H2 membrane receptors.

Inhibition of human primary melanoma cell proliferation by histamine is enhanced by interleukin-6.

BACKGROUND: Interleukin-6 (IL-6) is a bifunctional growth factor in malignant melanoma; its expression increases during the malignant progression of the disease. Histamine, detected in large amounts in normal and pathological proliferating tissues, is an important paracrine and autocrine regulator of normal and tumour cell proliferation as well. MATERIALS AND METHODS: We investigated the presence and function of IL-6 and histamine in the WM35 primary human melanoma cell line with respect to their direct role in cell proliferation and their regulatory interactions. RESULTS: IL-6 inhibited the proliferation of WM35 melanoma cells and increased significantly the expression of histidine decarboxylase as well as histamine production. It had dose-dependent effects on the proliferation: high concentration (10-5 M) was inhibitory through H1 histamine receptors while low histamine concentration acting on H2 receptors, with a simultaneous increase of cAMP, enhanced colony formation in the monolayer. Furthermore, IL-6 increased the H1- but decreased the H2-histamine receptor expression of the melanoma cells. On the other hand, histamine was locally synthesized by the WM35 melanoma cells. CONCLUSION: We suggest that the growth arrest induced by IL-6 is in part mediated by its dual action on histamine: a shift toward H1 receptor predominance and an elevation of locally produced histamine with prevalent action on the inhibitory response triggered through the H1 receptor. These findings suggest a local cross-talk between histamine and IL-6 in the regulation of melanoma growth.