Background pathology in BDF1 mice allowed to live out their life-span

Fifty male and 50 female BDF1 mice were observed allowing them to live out their life-span. Mortality up to 104 weeks of age was higher in males (42%) than in females (34%), and the 50% survival age was 112 weeks for males and 118 weeks for females. Body weight reached the peak at 82 weeks of age in males and 92 weeks of age in females, showing the mean body weight of 54.3 g for males and 48.0 g for females. The incidence of calculi and proteinaceous casts in the kidneys, that were not associated with exposure to chloroform, cell-alteration in the adrenal cortex, and islet cell hyperplasia in the pancreas was significantly higher in males than in females. On the other hand, hyaline droplet degeneration of the renal tubular epithelium, spindle cell proliferation in the adrenal cortex and milk-retention in the mammary glands occurred at a significantly higher incidence in females than in males. Cerebral mineralization in both sexes, atrophy and calcification of the testes and enlargement of the seminal vesicles of males, as well as cyst-formations in the ovary and endometrium of females developed at a very high incidence. Frequent neoplasms in males were hepatocellular adenomas and carcinomas, blood vessel tumours, pulmonary adenomas and carcinomas, and malignant lymphomas. In females, malignant lymphomas were the most common neoplasm, followed by blood vessel tumours, chromophobe pituitary adenomas and hepatocellular adenomas. Hepatocellular carcinomas developed only in males, whereas the histiocytic and lymphocytic types of malignant lymphomas and chromophobe cell adenomas arose solely or at a significantly higher incidence in females than in males.     

No direct correlation between mutant frequencies and cancer incidence induced by MeIQ in various organs of Big Blue® mice

The carcinogenicity of 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) was examined in Big Blue® female mice with the genetic background of C57BL/6N. With the administration of 300 ppm of MeIQ in their diet for 92 weeks, the Big Blue® female mice developed intestinal tumors and hepatocellular carcinomas. The incidences of adenocarcinomas were 42% (8/19) in the colon and 68% (13/19) in the cecum. The incidence of hepatocellular carcinomas was 84% (16/19). No carcinomas of the intestine or the liver were induced in the control group. As we previously reported, administration of 300 ppm of MeIQ in a diet for 12 weeks induced lacI mutants at the highest frequency in colonocytes, and at only less than one-tenth of the colon in cells of the liver, forestomach and bone marrow, indicating no direct correlation between the lacI mutant frequency (MF) and cancer incidence (CI). The fate of cells with lacI mutation in each organ should be taken into consideration to validate MF as an indicator of carcinogenic potency of a chemical in different organs.     

Carcinogenicities of heterocyclic amines in cooked food

Mutagenic heterocyclic amines in cooked foods were carcinogenic to mice, rats and/or monkeys, when they were given orally continuously. The most common target organ was the liver, but in CDF1 mice lung tumors, forestomach tumors, lymphomas/leukemias, and blood vessel tumors in the brown adipose tissues were also induced. In F344 rats, in addition to liver tumors, tumors in the Zymbal gland, skin, clitoral gland, small and large intestines, oral cavity, and mammary gland were also induced. Monkeys given IQ developed metastatic hepatocellular carcinomas.     

Genetic alterations in the Catnb gene but not the H-ras gene in hepatocellular neoplasms and hepatoblastomas of B6C3F(1) mice following exposure to diethanolamine for 2 years

The present study characterized the immunohistochemical localization of beta-catenin protein in hepatocellular neoplasms and hepatoblastomas in B6C3F(1) mice exposed to diethanolamine (DEA) for 2 years and evaluated genetic alterations in the Catnb and H-ras genes which are known to play important roles in the pathogenesis of liver malignancies. Genomic DNA was isolated from paraffin sections of each liver tumor. Catnb exon 2 (corresponds to exon 3 in human) genetic alterations were identified in 18/18 (100%) hepatoblastomas from DEA exposed mice. Deletion mutations (15/18, 83%) were identified more frequently than point mutations (6/18, 33%) in hepatoblastomas. Eleven of 34 (32%) hepatocellular adenomas and carcinomas from DEA treated mice had mutations in exon 2 of the beta-catenin gene, while only 1 of 10 spontaneous neoplasms had a deletion mutation of codon 5-6. Common to all liver neoplasms (hepatocellular adenomas, carcinomas and hepatoblastomas) was membrane staining for the beta-catenin protein, while cytoplasmic and nuclear staining was observed only in hepatoblastomas. The lack of H-ras mutations in hepatocellular neoplasms and hepatoblastomas suggests that the ras signal transduction pathway is not involved in the development of liver tumors following DEA exposure which is different from that of spontaneous liver tumors that often contain H-ras mutations.     

Mammary tumorigenesis in feral Mus cervicolor popaeus.

A pedigreed breeding population of feral Mus cervicolor popaeus with a high incidence of mammary tumors, arising between 6 and 14 months of age, is described. These mice were chronically infected with a type B retrovirus which is distantly related to the mouse mammary tumor virus (MMTV) of inbred strains of Mus musculus. MMTV-induced mammary tumors in inbred mice frequently (80%) contained an insertion of the viral genome into the int-1 or int-2 loci of the tumor cellular genome. These two cellular genetic loci were also altered by viral insertion in 11 of 20 M. cervicolor popaeus mammary tumor cellular DNAs tested. Results of our study of mammary tumorigenesis in feral mice demonstrate that viral-induced rearrangement and activation of the int loci are not limited to the genetic background of inbred mice selected for highly infectious MMTV and a high incidence of mammary tumors.     

DNA and S-phase distribution and incidence of metastasis in human primary lung carcinoma

The aim of the study was to investigate the relationship between DNA and S-phase distribution in primary non-small-cell lung carcinomas with the incidence of metastasis. Patients with non-small-cell lung carcinomas were divided into two groups depending on whether at time of surgery there were metastases or not, and these groups were correlated with the data obtained by flow cytometry or autoradiography. As expected from other studies, survival time was significantly longer for those patients without metastases at time of surgery (P = .0002) and the incidence of metastasis was significantly higher when the primary tumor was greater than or equal to 70 cm3 (P = .026). In this study, a total of 185 fresh specimens of lung carcinomas were investigated by flow cytometry. Patients with aneuploid tumors had a higher tendency to have metastases (P = .016). Patients with tumors with a higher proportion of S-phase cells measured by either flow cytometry or autoradiography demonstrated significant increase in the formation of metastases (P = .02 and P = .05). We feel that these results warrant further investigation with other primary tumors. A comparison of primary tumors that are known to rapidly metastasize vs. those that either slowly or rarely metastasize may prove to yield valuable insight into the important factors associated with metastatic potential.     

A multi-mineral natural product inhibits liver tumor formation in C57BL/6 mice

C57BL/6 mice were maintained for up to 18 months on high-fat and low-fat diets with or without a multi-mineral supplement derived from the skeletal remains of the red marine algae Lithothamnion calcareum. Numerous grossly observable liver masses were visible in animals on the "western-style" high-fat diet sacrificed at 12 and 18 months. The majority of the masses were in male mice (20 out of 100 males versus 3 out of 100 females; p = 0.0002). There were more liver masses in animals on the high-fat diet than on the low-fat diet (15 out of 50 on high-fat versus 5 out of 50 on low-fat; p = 0.0254). The multi-mineral supplement reduced the number of liver masses in mice on both diets (3 out of 25 male mice in the low-fat diet group without the supplement versus 1 out of 25 mice with supplement; 12 of 25 male mice in the high-fat diet group without the supplement versus 3 of 25 mice with supplement [p = 0.0129]). Histological evaluation revealed a total of 17 neoplastic lesions (9 adenomas and 8 hepatocellular carcinomas), and 18 pre-neoplastic lesions. Out of eight hepatocellular carcinomas, seven were found in unsupplemented diet groups. Steatosis was widely observed in livers with and without grossly observable masses, but the multi-mineral supplement had no effect on the incidence of steatosis or its severity. Taken together, these findings suggest that a multi-mineral-rich natural product can protect mice against neoplastic and pre-neoplastic proliferative liver lesions that may develop in the face of steatosis.     

Monitoring liver alterations during hepatic tumorigenesis by NMR profiling and pattern recognition

Human hepatocellular carcinoma (HCC) is the most recurrent malignancy of the liver and represents one of the main causes of cancer death worldwide. Furthermore, the liver is the most frequent site of metastatic colonization, and hepatic metastases are far more common than primary cancers in Western countries. A possible way of investigating liver diseases is to study the tissue metabolic profiles. High-resolution nuclear magnetic resonance (NMR) spectroscopy of hepatic tissue extracts was combined with pattern-recognition and visualization techniques to uncover metabolic differences among analyzed tissue types. Extracts were from primary HCC, chronic hepatitis-C-virus related cirrhotic tissues, hepatic metastases from colorectal carcinomas, and non-cirrhotic normal liver tissues adjacent to metastases as controls. We identified all metabolites present in the NMR spectra, and after statistical evaluation of all spectral classes, we were able to define the metabolic changes underlying the different liver conditions and diseases. In particular, the lactate and the glucose tissue signals were found to primarily discriminate the different histological samples. We followed the biochemical changes of human hepatic lesions through primary (HCC) and secondary (metastases from colorectal carcinoma) liver tumors, cirrhotic tissues, and non-cirrhotic histologically-confirmed normal liver tissues adjacent to metastases, achieving a metabolic differentiation of the various pathological states based upon the variation of the intracellular lactate/glucose ratio. It is suggested that such a signal pattern may act as a potential marker for assessing pathological hepatic lesions.     

Fhit Expression Protects Against HER2-Driven Breast tumor Development: Unraveling the Molecular Interconnections

The tumor suppressor gene FHIT is inactivated by genetic and epigenetic changes, i.e., loss of heterozygosity or promoter hypermethylation, in common human cancers. We recently showed that Fhit protein levels can be regulated by Fhit proteasome degradation mediated by EGF-dependent activation of EGFR family members, including HER2, whose overexpression is linked to poor prognosis in breast cancer. Analysis of a series of 384 human primary breast carcinomas revealed low/absent Fhit protein levels more frequently in HER2-overexpressing tumors. To test for a possible complementation of the FHIT and HER2 genes, tumor incidence was assessed in mice carrying one inactivated Fhit allele (Fhit+/-) crossed with FVB/N mice carrying the rat HER2/neu proto-oncogene driven by the mouse mammary tumor virus promoter. All Fhit heterozygous mice developed mammary tumors, whereas when both Fhit alleles (Fhit+/+) were present, tumor incidence was reduced in 27% of the mice, which remained tumor-free at 20 months. These findings suggest a protective role for FHIT in HER2-driven mammary tumors. Together, these data argue for the cooperation between Fhit and HER2 in breast carcinogenesis.     

Biomarkers of Residual Disease,Disseminated Tumor Cells,and Metastases in the MMTV-PyMT Breast Cancer Model

Cancer metastases arise in part from disseminated tumor cells originating from the primary tumor and from residual disease persisting after therapy. The identification of biomarkers on micro-metastases, disseminated tumors, and residual disease may yield novel tools for early detection and treatment of these disease states prior to their development into metastases and recurrent tumors. Here we describe the molecular profiling of disseminated tumor cells in lungs, lung metastases, and residual tumor cells in the MMTV-PyMT breast cancer model. MMTV-PyMT mice were bred with actin-GFP mice, and focal hyperplastic lesions from pubertal MMTV-PyMT;actin-GFP mice were orthotopically transplanted into FVB/n mice to track single tumor foci. Tumor-bearing mice were treated with TAC chemotherapy (docetaxel, doxorubicin, cyclophosphamide), and residual and relapsed tumor cells were sorted and profiled by mRNA microarray analysis. Data analysis revealed enrichment of the Jak/Stat pathway, Notch pathway, and epigenetic regulators in residual tumors. Stat1 was significantly up-regulated in a DNA-damage-resistant population of residual tumor cells, and a pre-existing Stat1 sub-population was identified in untreated tumors. Tumor cells from adenomas, carcinomas, lung disseminated tumor cells, and lung metastases were also sorted from MMTV-PyMT transplant mice and profiled by mRNA microarray. Whereas disseminated tumors cells appeared similar to carcinoma cells at the mRNA level, lung metastases were genotypically very different from disseminated cells and primary tumors. Lung metastases were enriched for a number of chromatin-modifying genes and stem cell-associated genes. Histone analysis of H3K4 and H3K9 suggested that lung metastases had been reprogrammed during malignant progression. These data identify novel biomarkers of residual tumor cells and disseminated tumor cells and implicate pathways that may mediate metastasis formation and tumor relapse after therapy.     

Histogenesis of hyperplasia and carcinomas of the liver arising around central veins in mice ingesting chlorinated hydrocarbons.

The development of hyperplastic and neoplastic lesions of parenchymal cells of the liver adjacent to central veins was observed in C3H mice ingesting the chlorinated hydrocarbons, dieldrin or aldrin, in the diet. Lesions could be followed from pericentral hyperplasia to areas of hyperplasia, nodules of hyperplasia, small hepatocellular carcinomas, and large well-developed carcinomas, occasionally with metastases. Sometimes pericentral hyperplasia was diffuse throughout most or all of one lobe of the liver. These hyperplastic cells collided to become one large nodule and also one large carcinoma. The carcinomas were well-differentiated or moderately well-differentiated and grew on transplantation to isologous hosts. Histologically, the hyperplastic cells adjacent to central veins were increased in size, frequently with double nuclei. Carcinoma cells varied in size and shape and were huge with large nuclei, prominent nucleoli, and eosinophilic cytoplasm. Similar hepatocellular carcinomas were seen previously with carbon tetrachloride, another organochlorine chemical.     

Characterization of four in vitro established canine mammary carcinoma and one atypical benign mixed tumor cell lines

Summary Five spontaneous canine mammary tumors were cultured in vitro and cell lines were established. The tumors included three frozen carcinomas, fine-needle aspirate from one fresh carcinoma, and one fresh atypical benign mixed tumor. The cell lines have so far been cultured for about 2 yr and passaged between 45 and 200 times. The cell lines expressed different types of intermediate filaments, including a heterogenous pattern. In some cases no intermediate filaments were expressed. Ultrastructure studies showed epithelial cells and cells intermediate between epithelial and myoepithelial types. Retrovirus associated A-particles were found in two carcinomas. The mixed mammary tumor cell line formed ductlike structures in collagen substrate. The cell lines grew when inoculated s.c. into male nude mice. Two carcinomas caused lymph node metastases in two mice and another carcinoma single lung metastases in one tested mouse. DNA hypodiploidy, studied by flow cytometry, in one of the primary carcinoma was retained in vitro, and this cell line showed polyploidy during later passages. The other cell lines had a more unstable DNA profile, although a tendency for polyploidy was found. These findings were also illustrated in chromosome studies.     

Promoting effect of ovariectomy on hepatocellular tumorigenesis induced in mice by 3'-methyl-4-dimethylaminoazobenzene.

The treatment of female C57BL/6 x DS-F1 mice with 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB) at 10, 12, 14, 16 and 18 days of age resulted in the development of hepatocellular adenomatous nodules after 10 months of age. Ovariectomy in these mice at 1 month of age hastened the development of adenomatous nodules, which then first appeared at 8 months of age. The incidence of adenomatous nodules in females ovariectomized at the age of 1 month was much higher than that in intact females of the same age. These results showed that the ovaries exerted a suppressive effect on the development of adenomatous nodules. To determine the time from which the ovaries exert this suppressive effect, females were ovariectomized at 4, 6, 8, and 10 months of age, and the incidences of adenomatous nodules were compared at 10 and 12 months of age. Delayed ovariectomy after 8 months of age did decrease the incidence of adenomatous nodules at 10 and 12 months of age, but ovariectomy after 4 and 6 months of age did not. When the incidence of adenomatous nodules in females ovariectomized at 10 months of age was examined over the subsequent 6 months, it became significantly higher after 14 months of age compared with that in intact females. The results show that the ovariectomy has the promoting effect on the development of adenomatous nodules in the liver induced by 3'-Me-DAB after 6 months of age.     

Fas-negative osteosarcoma tumor cells are selected during metastasis to the lungs: the role of the Fas pathway in the metastatic process of osteosarcoma

Low expression of Fas by different tumors including osteosarcoma, correlates with poor prognosis. We found that osteosarcoma lung metastases from patients expressed negligible amounts of Fas, but primary tumors often expressed high Fas levels. The reason for this discrepancy is unknown. We hypothesized that because FasL is constitutively expressed in the lungs, Fas-positive (Fas(+)) tumor cells entering the lungs would bind with FasL and die from Fas-induced apoptosis, resulting in the "selection" of Fas-negative (Fas(-)) cells, which would eventually form metastases. To test this hypothesis, we injected K7 osteosarcoma cells, which express functional Fas in vitro, into mice and confirmed that its bone tumors were Fas(+), but lung metastases were Fas(-). Next, to inhibit Fas signaling without affecting Fas expression, we transfected these cells with a FADD-dominant negative (FDN) plasmid and developed K7/FDN cells. Metastases formed by K7/FDN cells contained Fas(+) tumor cells. Moreover, K7/FDN cells were retained in the lungs longer and formed more lung metastases than K7 cells. In addition, the incidence of lung metastases in FasL-deficient mice injected with K7 cells was higher than that in wild-type mice. Metastases from FasL-deficient mice but not from wild-type mice contained Fas(+) tumor cells. Based on that, we conclude that Fas(-) osteosarcoma cells are selected during lung metastases formation and that inhibition of Fas signaling in tumors or lack of FasL in the host environment allows the proliferation of Fas(+) osteosarcoma cells in the lungs and promotes metastases growth. Therefore, Fas may be considered as a new therapeutic target for osteosarcoma treatment.     

Bulbourethral (Cowper's) gland abnormalities in inbred laboratory mice

Abnormalities of the bulbourethral (Cowper's) glands are rare in most strains of inbred laboratory mice. Since the glands are covered with striated muscle and because of their small size, the bulbourethral glands are often overlooked at necropsy. We review the gross and microscopic anatomy of the bulbourethral glands in laboratory mice and report in SJL/J and RBF/DnJ mice a series of anomalies of this gland that appear to be common in these strains and relatively strain specific. From 14 different inbred strains of mice that had been submitted for necropsy over a period of 4.5 years for various clinical complaints, including enlargement of the perianal region, 23.8% of SJL/J mice (27/113) and 51.2% of RBF/DnJ mice (21/41) had cystic hyperplasia of the bulbourethal glands. Furthermore 7.3% of BALB/cByJ mice (3/41) had cystic glands and 6.7% of DBA/2J mice (4/60) had enlarged bulbourethal glands, three of which (5.0%) were abscessed. All selected mice were active male breeders older than 6 months of age from production colonies at The Jackson Laboratory. Detailed examination of bulbourethral glands of retired SJL/J breeders revealed cystic glands in 83.8% (109/130) of mice. No abnormalities were found in the other inbred strains of mice investigated.     

Fine needle aspiration of the liver. Significance of hepatocytic naked nuclei in the diagnosis of hepatocellular carcinoma

Fine needle aspiration (FNA) smears from 60 cases of histologically (34) or clinically (26) confirmed hepatocellular carcinomas were reviewed. In about 90% of the cases, the cytologic preparations contained clusters of malignant cells with variable degrees of hepatocytic features and a distinctive type of naked nuclei. These naked nuclei had features similar to those of the malignant hepatocytes, but with more evident atypia. They were numerous in about 75% of the cases and less frequent in 15%; in three cases, they were practically absent. All three cases of well-differentiated hepatocellular carcinomas presented with numerous naked nuclei with slight atypia. Flowerlike-configured naked nuclei were especially found in poorly differentiated hepatocellular tumors. Hepatocytic naked nuclei were rarely found in FNA smears from normal liver, metastases, cysts or degenerative, regenerative and inflammatory liver processes; when seen in these cases, they showed no atypia. The presence of atypical hepatocytic naked nuclei appears to be a very useful criterion for the diagnosis of hepatocellular carcinoma.     

Hepatitis a virus infection of HBsAg-producing hepatocellular carcinomas in athymic mice

Summary Two human hepatocellular carcinoma cell lines were grown in athymic mice. Morphologically, the tumors resembled hepatocellular carcinomas. HBsAg, anti-HBs, and -fetoprotein were detected in sera of mice bearing tumors of PLC/PRF/5 or Hep 3B cells; their amounts correlated with tumor weight. Tumors of both cell lines were infected with cell culture-adapted hepatitis A virus (HAV); HAV antigen and infectious virus were recovered from infected tumors of PLC/PRF/5 and Hep 3B cells. Our results indicate that HAV-infected tumors may be useful in studying the production in vivo of hepatitis A virus antigen.     

Expression of inducible nitric oxide (NO) synthase but not prevention by its gene ablation of hepatocarcinogenesis with fibrosis caused by a choline-deficient, L-amino acid-defined diet in rats and mice.

Expression of inducible nitric oxide synthase (iNOS) and effects of iNOS gene ablation on the hepatocarcinogenesis associated with fibrosis caused by a choline-deficient, L-amino acid-defined (CDAA) diet, were examined in male F344 rats and C57BL/6J wild-type and iNOS-/- mice. Western blot, RT-PCR and immunohistochemical analyses revealed increased expression of iNOS protein and mRNA in the livers of rats and wild-type mice fed a CDAA diet for 12-80 weeks, associated with elevated serum NO(x) and liver nitrotyrosine levels. iNOS-/- mice demonstrated greater liver injury and fibrosis in the early stage than their wild-type counterparts, but this did not significantly affect the incidence and multiplicity of altered foci, adenomas and hepatocellular carcinomas in spite of immunohistochemical iNOS expression in these lesions. Results suggested no major determinant roles of the expressed iNOS in the development of liver tumors caused by the CDAA diet.     

Behavior of metastatic and nonmetastatic breast tumors in old mice

Breast cancer incidence and mortality increase with age. A better understanding of the biological behavior of metastatic and nonmetastatic breast tumors in older subjects may help to develop improved breast cancer therapies. In this study, we used syngeneic metastatic (4TO7cg) and nonmetastatic (64pT) mouse breast tumor models at three age levels to evaluate various characteristics that are considered to be important for effective anti-breast cancer immunotherapy. These included tumor size and growth, metastases, vascularization, gene expression levels of the tumor-associated antigen (TAA) Mage-b (homologous to human MAGE-B) in primary breast tumors and metastases, and the presence of CD4(+) and CD8(+) T cells in the inguinal lymph nodes at the site of the tumor. The primary breast tumors and metastases were generated by injection of mouse mammary tumor cell lines 4TO7cg or 64pT into a mammary fat pad of normal 3-, 9-, or 21/24-month old BALB/c mice. In the nonmetastatic breast tumor model, significantly smaller tumors were observed in old compared with young mice. This was associated with a significant increase in the percentage of CD8(+) T cells in inguinal lymph nodes and significantly higher Mage-b expression levels in the primary tumors at old age. In the metastatic (4TO7cg) breast tumor model, a less pronounced, not statistically significant, smaller tumor size was found in the old mice, without a difference in the percentage of CD8(+) T cells or Mage-b expression levels. However, in this mouse model almost all metastases showed high levels of Mage-b expression (2- to 3-fold higher than the primary tumors in the same animals) regardless of age. These results indicate that the metastatic and nonmetastatic breast tumor models could be useful model systems to analyze how breast cancer vaccines for humans can be tailored to old age.     

Influence of age and sex on carcinoma and cirrhosis of the liver in AXC strain rats ingesting 0.025% N-2-fluorenyldiacetamide.

Inbred A X C male and female rats, 4, 12, 24, and 52 weeks of age, ingested 0.025% N-2-fluorenyldiacetamide in a semisynthetic diet. Both age and sex were important in the development of hepatic lesions. The 4- and 12-week-old males developed a higher incidence of carcinomas and cirrhosis of the liver than did the females of the same ages or the males and females 24 and 52 weeks of age. Four-week-old male rats had more carcinomas per liver, larger carcinomas, more poorly differentiated (as compared with well differentiated), and some carcinomas were cholangiocellular as well as hepatocellular. There were a few hepatic lesions in the younger female rats; however, female rats of all ages were relatively resistant to hepatic carcinogenesis.