Transgenic TIMP-1 inhibits simian virus 40 T antigen-induced hepatocarcinogenesis by impairment of hepatocellular proliferation and tumor angiogenesis.

Tissue inhibitors of metalloproteinases (TIMP) block proteolytic degradation of extracellular matrix and consequently impede tumor invasion and metastasis. In addition, we have previously reported that hepatic TIMP-1 modulation alters the susceptibility of the liver to oncogene (simian virus 40 T-antigen; TAg)-induced tumorigenesis in a double-transgenic mouse model. To identify the cellular processes by which TIMP-1 inhibits hepatocarcinogenesis, we examined the effects of TIMP-1 on four specific events that are important during tumorigenesis: hepatocellular proliferation, apoptosis, the stromal characteristics of the liver, and tumor vascularization. Transgenic mice with elevated or reduced hepatic TIMP-1 expression were bred independently with TAg transgenics. Liver tissue from littermates were analyzed by in situ hybridization with TIMP-1 cDNA probes; gelatin enzymography; immunohistochemistry for proliferating cell nuclear antigen, von Willebrand factor, and collagen type IV; reticulin histochemistry; and collagens type III and IV, laminin, fibronectin, and CD31 immunoblotting. We demonstrate that TIMP-1 overexpression significantly inhibited the proliferation of hepatocytes in TAg mice but did not affect their apoptotic index, the hepatic parenchymal architecture, or extracellular matrix composition, including collagens type III and IV, laminin, and fibronectin. Moreover, the hepatocellular carcinomas formed in TIMP-1-overexpressing mice had significantly reduced tumor vascularization; conversely, tumor vascularization was significantly increased in TIMP-1-reduced livers. These data indicate that TIMP-1 inhibits TAg-induced hepatocarcinogenesis by altering hepatocellular proliferation and tumor vascularization, without any effect on hepatocyte apoptosis and stromal composition. To our knowledge, this is the first in vivo demonstration that genetic modulation of TIMP-1 inhibits cellular proliferation and angiogenesis during hepatocarcinogenesis. This potentially extends the use of matrix metalloproteinase inhibitors in cancer beyond control of invasion and metastasis.

Genetic background influences timing, morphology and dissemination of lymphomas in p53-deficient mice.

To examine the influence of genetic background on tumorigenesis in p53-deficient mice, we used selective breeding to produce congenic mice with a null p53 gene mutation introduced into the VM inbred strain. Cohorts of homozygous p53 null (-/-) mice from the original C57B6/129Sv mixed strain and the VM congenic strain were monitored for spontaneous tumor development, as were control cohorts of wild-type (+/+) and heterozygous (+/-) animals. Twenty-six of 28 C57B6/129Sv (-/-) mice died by the study end date (median survival =184.5 days). Twenty-three of 26 VM (-/-) mice died and their survival was significantly shorter (111 days, P<0.0001). Of 26 C57B6/129Sv (-/-) mice that died, 21 were autopsied: all 21 had lymphomas. Of 26 VM mice that died (23 -/-, 3 +/-), 21 were autopsied: 19 developed lymphoma and two had sarcomas. Several mice had additional neoplasms. Lymphomas in VM mice were distinct from those in C57B6/129Sv mice in that they i) arose on average more than two months earlier, ii) involved thymus more often than spleen or lymph nodes and iii) were more often poorly differentiated, high grade tumors. These results demonstrate that genetic background alone influences the onset, morphology and dissemination of lymphomas in p53-deficient mice and suggest the presence of genes which modify the timing and biological nature of lymphomas in these mice.

Host TIMP-1 overexpression confers resistance to experimental brain metastasis of a fibrosarcoma cell line.

Within the tumor-stromal microenvironment a disrupted balance between matrix metalloproteinases (MMPs) and their inhibitors compromises the integrity of the extracellular matrix and promotes malignancy. Tissue inhibitors of metalloproteinases (TIMPs) have been linked to tumor suppression in studies of genetically altered tissue culture cells and in analyses of clinical specimens in situ. We generated transgenic mice as a model system to test the relationship between TIMP-1 levels in a host organ and susceptibility to experimentally targeted metastasis. Ectopically overexpressed TIMP-1 in the brain resulted in a tissue microenvironment with elevated protein levels of this natural MMP inhibitor. Metastatic challenge provided by lacZ-tagged fibrosarcoma cells permitted high-resolution analysis of metastatic load and pattern. We found that elevated host TIMP-1 imposed resistance to experimental metastasis of fibrosarcoma: In TIMP-1 overexpressing mice, brain metastases were significantly reduced by 75% compared to wild-type littermates. Our findings demonstrate that ectopic TIMP-1 expression efficiently exerts a suppressive effect on metastasizing tumor cells.

Human metastatic prostate PC3 cell lines degrade bone using matrix metalloproteinases.

Bone metastases are often associated with osteolysis and subsequent pathological fractures. To determine if metastatic human cancer cells can directly degrade non-mineralized and mineralized bone, we used prostate PC3 adenocarcinoma cell lines, which were originally established from skeletal metastases. We show that PC3 cells and their conditioned medium degraded non-mineralized, osteoid-like radiolabelled extracellular matrices from human Saos2 and U2OS osteoblast-like cells. These cells also directly degraded mineralized bone by inducing (45)Ca release from rat fetal calvariae and forming resorption pits on bone slices, an effect increased by transforming growth factor-beta(1). A role for matrix metalloproteinases in degradation was shown by: (1) stimulation by the phorbol ester TPA of PC3-induced matrix degradation and release of matrix metalloproteinase activity; (2) abrogation of matrix degradation by 1,10-phenanthroline, a metalloproteinase inhibitor, and (3) degradation of purified type I collagen by PC3 cells and their conditioned medium. We demonstrate that human prostate cancer cells can directly degrade bone-related matrices and that matrix metalloproteinases have a role in this process.

Direct osteolysis induced by metastatic murine melanoma cells: role of matrix metalloproteinases.

We examined the osteolytic ability of metastatic cells and the role of tumour matrix metalloproteinases (MMPs) in bone degradation. The histomorphometry of experimental bone metastases of B16/F1 melanoma cells showed that osteolysis was associated with a 90% decrease in osteoclast number and predominance of cancer cells overlaying resorption pits. In vitro, B16/F1 cells and their conditioned medium (CM) degraded 3H-proline-labelled extracellular matrices from osteoblast-like cells and 45Ca-labelled calvariae. Using bone slices, we observed morphological evidence of degradation by B16/F1 cells. A role for tumour MMPs in bone degradation was supported by inhibition of degradation by 1,10-phenanthroline, collagen I degradation by tumour cells and the presence of TPA-inducible M(r) 90,000, 84,000 and 64,000 gelatinolytic, and 54,000 caseinolytic bands in B16/F1-CM. These studies indicate that metastatic cancer cells degrade bone matrix directly and that this is partially mediated by MMPs.

Inhibition of SV40 T antigen-induced hepatocellular carcinoma in TIMP-1 transgenic mice.

The potential of tissue inhibitors of metalloproteinases (TIMPs) to inhibit neoplastic progression has been postulated from studies of genetically manipulated cells. To investigate whether the TIMP-1 expressed in a host tissue suppresses cancer in vivo and to identify the affected stages, we developed transgenic mice with constitutive overexpression or reduction of TIMP-1 in the liver. In double transgenic experiments, the TIMP-1 lines were crossed with a second transgenic line which expresses the Simian Virus 40t/T antigen (TAg). This viral oncogene leads to heritable development of hepatocellular carcinomas with a 100% incidence. Effects of TIMP-1 coexpression on the TAg-induced neoplasms were determined at the tissue and cellular level. Here, we report that overexpression of hepatic TIMP-1 blocked the development of TAg-induced hepatocellular carcinomas. High TIMP-1 levels inhibited not only the later stages in tumor development (growth and angiogenesis), but also events associated with tumor initiation (altered hepatocyte cytology and tissue architecture). We further show that an antisense-mediated reduction of TIMP-1 resulted in a more rapid tumor initiation and progression. These data demonstrate that intrinsic TIMP-1 levels contribute to a tissue's susceptibility to viral oncogene-induced tumorigenesis.

Tumor-bone interactions in skeletal metastasis.

Bone metastases are a frequent clinical problem in patients with breast, prostate, and other cancers. Formation of these lesions is a site-specific process determined by multiple cellular and molecular interactions between the cancer cells and the bone microenvironment. Clinical studies, and in vivo and in vitro experimental approaches, have been useful to dissect different stages of this process. Mechanisms identified as relevant to cancer spreading and tumoral growth in the bones include (a) early vascular spread of cancer cells to bones; (b) adhesion of cancer cells to the bone microvasculature and matrix components; (c) presence of growth factors and chemo-attractants in bone; (d) osteolysis by osteoclasts, tumor associated macrophages, and cancer cells; and (e) tumor-induced local osteoblastic proliferation. Although none of these mechanisms alone are responsible for the development of bone metastases, their investigation may lead to novel therapeutic approaches that specifically block these stages and, thus, may hinder development of bone metastasis. The use of bisphosphonates and other experimental strategies already is being tested in clinical trials.

Human chromosomes: evaluation of processing techniques for scanning electron microscopy.

Methods for scanning electron microscopy (SEM) of chromosomes have been developed in the last two decades. Technical limitations in the study of human chromosomes, however, have hindered the routine use of SEM in clinical and experimental human cytogenetics. We compared different methodologies, including metal impregnation, air drying and specimen coating. SEM preparation of human chromosomes in which osmium impregnation is mediated by tannic acid, yielded more reproducible results when compared with osmium impregnation protocols previously described. The level of osmium impregnation was systematically evaluated by imaging chromosomes in the backscattering mode. Critical point drying and a light gold-palladium coating were essential for appropriate secondary electron imaging of chromosomes. With this method, and in a preliminary quantitative analysis, we show that our SEM technique is more sensitive than light microscopy for the detection of aphidicolin-induced fragile sites. This technical approach is useful for chromosomal studies requiring resolution higher than that obtained by light microscopy. Also, it allows the use of clinical and archival chromosomal samples prepared by routine cytogenetic techniques.

Mechanisms involved in the metastasis of cancer to bone.

The metastasis of cancer to bone is a frequent outcome of common malignancies and is often associated with significant morbidity due to osteolysis. Bone metastasis is also selective in that a disproportionately small number of malignancies account for the majority of tumors which spread to bone. While the mechanisms of bone destruction have been studied, those responsible for the site-specific nature of bone metastasis are poorly understood. As a metastatic target, bone is unique in that it is continuously being remodelled under the influence of local and systemic growth factors, many of which are embedded in the bone matrix. This review summarizes evidence for the hypothesis that the formation of metastatic tumors in bone is the consequence of a unique microenvironment where metastatic cells can alter the metabolism of bone, thereby regulating the release of soluble bone-derived growth factors as a consequence of bone resorption. These, in turn, can modulate the malignant phenotypic properties of receptive cells. Transforming growth factor-beta is one factor which can promote the growth and motility of Walker 256 cells, a rat cell line with a propensity to metastasize spontaneously to bone.