Rodent Cancer Cells Research Articles

Visualization of GFP-expressing tumors and metastasis in vivo.

We have developed mouse models of metastatic cancer with genetically fluorescent tumors that can be imaged in fresh tissue, in situ, as well as externally. To achieve this capability, we have transduced the green fluorescent protein (GFP) gene, cloned from the bioluminescent jellyfish Aequorea victoria, into a series of human and rodent cancer cell lines that were selected in vitro to stably express GFP in vivo after transplantation to metastatic rodent models. Techniques were also developed for transduction of tumors by GFP in vivo. With this fluorescent tool, we detected and visualized for the first time tumors and metastasis in fresh viable tissue or in situ in host organs down to the single-cell level. GFP tumors on the colon, prostate, breast, brain, liver, lymph nodes, lung, pancreas, bone, and other organs can also be visualized externally, transcutaneously by quantitative whole-body fluorescence optical imaging. Real-time tumor and metastatic growth and angiogenesis and inhibition by representative drugs can be imaged and quantified for rapid antitumor, antimetastatic, and antiangiogenesis drug screening. The GFP-transfected tumor cells enabled a fundamental advance in the visualization of tumor growth and metastasis in real time in vivo.

A study of membrane activity in rat prostate cancer cells: an evaluation of the FM1-43 dye technique.

A study was initiated to test whether the FM1-43 dye technique could be applied to the study of endocytic membrane activity in two rodent prostate cancer (MAT-LyLu and AT-2) cell lines of markedly different metastatic ability. The lipophilic dye FM1-43, which has frequently been used to monitor endo/exocytic activity in excitable cells was employed. We found, as in excitable tissues, that both strongly metastatic (MAT-LyLu) and weakly metastatic (AT-2) cells in culture take up FM1-43 to give vesicular staining of a variable pattern, which appeared to differ between the two cell lines. However, unlike excitable tissues, neither cell line subsequently released the dye. Indeed, both cell lines retained the dye through several rounds of cell division suggesting that dye incorporated by cells does not enter the endo/exocytotic cycle. Uptake of dye was independent of temperature, Na+/K+ gradients, pH or metabolism. We suggest that passive accumulation of FM1-43 can occur in cancer cells and should not, automatically, be interpreted as evidence of endocytosis.

PEG-3, a nontransforming cancer progression gene, is a positive regulator of cancer aggressiveness and angiogenesis.

Cancer is a progressive disease culminating in acquisition of metastatic potential by a subset of evolving tumor cells. Generation of an adequate blood supply in tumors by production of new blood vessels, angiogenesis, is a defining element in this process. Although extensively investigated, the precise molecular events underlying tumor development, cancer progression, and angiogenesis remain unclear. Subtraction hybridization identified a genetic element, progression elevated gene-3 (PEG-3), whose expression directly correlates with cancer progression and acquisition of oncogenic potential by transformed rodent cells. We presently demonstrate that forced expression of PEG-3 in tumorigenic rodent cells, and in human cancer cells, increases their oncogenic potential in nude mice as reflected by a shorter tumor latency time and the production of larger tumors with increased vascularization. Moreover, inhibiting endogenous PEG-3 expression in progressed rodent cancer cells by stable expression of an antisense expression vector extinguishes the progressed cancer phenotype. Cancer aggressiveness of PEG-3 expressing rodent cells correlates directly with increased RNA transcription, elevated mRNA levels, and augmented secretion of vascular endothelial growth factor (VEGF). Furthermore, transient ectopic expression of PEG-3 transcriptionally activates VEGF in transformed rodent and human cancer cells. Taken together these data demonstrate that PEG-3 is a positive regulator of cancer aggressiveness, a process regulated by augmented VEGF production. These studies also support an association between expression of a single nontransforming cancer progression-inducing gene, PEG-3, and the processes of cancer aggressiveness and angiogenesis. In these contexts, PEG-3 may represent an important target molecule for developing cancer therapeutics and inhibitors of angiogenesis.

Inhibition of K- ras-transformed rodent and human cancer cell growth via induction of apoptosis by irreversible inhibitors of ras endoprotease

Proteolytic removal of the carboxyl terminal tripeptide of Ras oncoproteins is important in the Ras function. Two chloromethyl ketones, BFCCMK and UM96001, designed to be the Ras C-terminal sequence-specific endoprotease inhibitors, at low micromolar concentrations (5.0 μM), potently inhibit the growth of ras-transformed rodent and human cancer cells, whereas untransformed NIH/3T3 cells are not affected under the same conditions. Furthermore, BFCCMK and UM96001 block more than 98% of the anchorage-independent clonogenic growth of ras-transformed rat and human cancer cells at low micromolar concentrations. The blocking of cancer cell growth may be due to the selective induction of apoptosis of ras-transformed cells by these inhibitors. These results provide the first experimental evidence that the endoproteolysis of Ras oncoproteins is important for the growth and apoptosis of ras-transformed cancer cells. Therefore, the Ras C-terminal sequence-specific endoprotease may be a potential new target for the treatment of human cancers induced by ras mutations.

P53 tumor suppressor gene: from the basic research laboratory to the clinic--an abridged historical perspective.

Tumor suppressor genes maintain tissue homeostasis by controlling cellular proliferation, terminal differentiation and programmed cell death (1,2). The p53 tumor suppressor gene has come to the forefront of cancer research because it is commonly mutated in human cancer and the spectrum of p53 mutations in these cancers is providing clues to the etiology and molecular pathogenesis of cancer (3-8). Of the ~6.5 million cancer cases worldwide each year, 2.4 million tumors are estimated to contain a p53 mutation (examples shown in Figure 1). In the most common lethal types of cancers found in the US population, the estimate is over 300 000 cancers (Table I). These are necessarily crude estimates, because the mutation frequency differs among populations due to dissimilar exposures to environmental carcinogens (and perhaps other reasons such as genetic variation among ethnic groups of genes involved in critical biologic pathways), and selection bias might confound figures derived from early studies. Nevertheless, the high frequency of p53 mutations attests to their potential importance in the pathogenesis, diagnosis and treatment of human cancer. The 16 year history of p53 investigations is a paradigm in cancer research, illustrating the convergence of previously parallel lines of basic, clinical, and epidemiologic investigation and the rapid transfer of research findings from the laboratory to the clinic. This rich history of scientific accomplishment is briefly reviewed in Table n. The initial observations in 1979 of a cellular protein of ~53 kDa complexing with the large T antigen of SV-40 DNA virus, and of accumulation of p53 protein in the nuclei of neoplastic rodent cells stimulated several researchers to investigate the presence of p53 in tumors and its potential role in carcinogenesis. The p53 gene cloned from neoplastic rodent and human cells was then shown to have weak oncogenic activity. In the late 1980s, researchers discovered that they were studying p53 mutants instead of the wild-type gene; thus the first decade of p53 history can be confusing to the novice reader. Whereas many p53 mutants acted as a dominant-acting oncogene, the wild-type gene suppressed both the neoplastic transformation of rodent fibroblasts in vivo and the growth of rodent and human cancer cells in vitro and in vivo. To the surprise of cancer researchers in 1989, p53 was found to be mutated frequently in human cancers, and the search for p53 functions intensified which has resulted in an explosion of reports in the literature (Figure 2). Recent studies indicate that the p53 protein is involved in gene transcription, DNA synthesis and repair, senescence, genomic plasticity, and in programmed cell death (2-5,7,913). These complex biochemical processes are performed by multicomponent protein machines, so it is not surprising that the p53 protein forms complexes with other cellular proteins, and that oncoviral proteins of certain DNA viruses alter the functions of these protein machines by binding to p53 and perturbing its interaction with other cellular protein components (Figure 3). Ongoing studies are both defining the threedimensional structure of these p53-containing protein complexes and uncovering the regulation of their precise functions. p53 is clearly a component in a biochemical pathway(s) (5) central to human carcinogenesis; p53 protein alterations due to missense mutations and loss of p53 protein by nonsense or frameshift mutations provide a selective advantage for clonal

Differential expression of voltage-activated Na + currents in two prostatic tumour cell lines: contribution to invasiveness in vitro

The voltage-gated ionic currents of two rodent prostatic cancer cell lines were investigated using the whole-cell patch clamp technique. The highly metastatic Mat-Ly-Lu cells expressed a transient, inward Na + current (blocked by 600 nM tetrodotoxin), which was not found in any of the weakly metastatic AT-2 cells. Although both cell lines expressed a sustained, outward K + current, this occurred at a significantly higher density in the AT-2 than in the Mat-Ly-Lu cells. Incubation of the Mat-Ly-Lu cell line with 600 nM tetrodotoxin significantly reduced the invasive capacity of the cells in vitro. Under identical conditions, tetrodotoxin had no effect on the invasiveness of the AT-2 cells.

Analyzing the metastatic phenotype.

The dissemination of cells from a primary tumor, resulting in the progressive growth of metastatic carcinoma in distant sites, is the most common cause of death of cancer patients. The observations from clinical studies and the results of experimental studies using rodent tumors and human cancer cells implanted into immunodeficient host animals suggest that metastasis is not a random event, but rather the result of a sequence of selective events, many of which involve interactions with elements of the microenvironment of the primary and metastatic tumors. Analysis of the metastatic potential of a human tumor cell population has been greatly improved by the introduction of orthotopic models of tumor growth and metastasis, which have demonstrated that implanting human tumor cells into the appropriate tissue in an immunodeficient rodent can increase both tumor take and incidence of metastasis. These will be the models that should be used to validate the identity of candidate metastasis-associated genes, and to determine the value of new forms of therapy, either genetic or pharmacological, for controlling metastatic cancer growth.

VITAMIN-K-3 INDUCES CELL-DEATH VIA APOPTOSIS IN HUMAN CERVICAL-CARCINOMA TSGH8302 CELLS

Vitamin K-3 (VK3) exhibits antitumor activity in rodent and human cancer cells. The relationship between VK3-induced cytotoxicity, and morphological changes in human cervical carcinoma TSGH8302 cells were studied. Cell viability was analyzed by sulforhodamine B protein binding and clonogenic assays. Inhibition of cell growth by VK3 was cell density dependent as measured by IC50 values, which were 17 mu M at 0.5 x 10(4) cells/well and 36 mu M at 1.0 x 10(4) cells/well. Treatment of 10(6) cells with VK3 (5-100 mu M) for 1 h followed by recovery for 24 h caused depletion of the reduced glutathione pool. Under light, fluorescence and scanning electron microscopes, cells showed morphological changes after 1-h treatment with 25 mu M VK3, followed by a 4-h or 12-h recovery. The cells appeared retracted with blebs but no surface microvilli. They exhibited chromatin condensation and DNA fragmentation. Since these phenomena are characteristics of apoptosis, VK3-induced cell death appears to be mediated by apoptosis.

Vitamin K 3 Inhibits Growth of Human Hepatoma HepG2 Cells by Decreasing Activities of Both p34 cdc2 Kinase and Phosphatase

Synthetic vitamin K 3 is known to exhibit in vitro and in vivo antitumor activity against rodent and human cancer cells. To determine vitamin K 3′s effect on the cell cycle, we treated both synchronized and asynchronized human hepatoma HepG2 cells with 100 μM vitamin K 3 for 1 h and followed cells to recover for various times, then monitored cell cycle progression by flow cytometry and by phosphorylation status of p34 cdc2 kinase. Vitamin K 3 induced a delay at the S/G2 phase of the cell cycle. This was accompanied by constant hyperphosphorylation status and decreased activity of p34 cdc2 kinase. Protein-tyrosine phosphatase activity also decreased 2- to 3-fold after vitamin K 3 treatment. Our results suggest that vitamin K 3 inhibits the growth of HepG2 cells via a cell cycle progression delay and altered phosphorylation patterns and activities of both p34 cdc2 kinase and protein-tyrosine phosphatase.

6-Methyl-1,3,8-trichlorodibenzofuran (MCDF) is an antiestrogen in human and rodent cancer cell lines: Evidence for the role of the Ah receptor

6-Methyl-1,3,8-trichlorodibenzofuran (MCDF) is a relatively nontoxic analog of 2,3,7,8-tetrachlorodibenzo- p-dioxin. Treatment of aryl hydrocarbon (Ah)-responsive MCF-7 human breast cancer cells with 100 n m MCDF resulted in the inhibition of 17β-estradiol-induced proliferation and the secretion of the 34-, 52-, and 160-kDa proteins. After treatment of the cells with 17β-[ 3H]estradiol, 100 n m of MCDF caused a decrease in the accumulation of the radiolabeled nuclear estrogen receptor (ER) complex in these cells. In parallel experiments, the antiestrogenic effects of MCDF were also determined in Ah-responsive wild-type Hepa 1c1c7 cells and Ah-nonresponsive class 1 and class 2 mutant cells. Treatment of the wild-type cells with 17β-[ 3H]estradiol and 100 n m MCDF caused a decrease in the accumulation of radiolabeled nuclear ER complex in these cells whereas no significant effects were observed in the mutant cells as determined by velocity sedimentation analysis. Comparable results were obtained using ER antibodies to measure the decrease in immunoreactive nuclear ER. In addition, both actinomycin D and cycloheximide inhibited the MCDF-mediated decrease of nuclear ER levels in the Hepa 1c1c7 wild-type cells. Although 100 n m MCDF did not induce cytochrome P-450-dependent monooxygenases in the MCF-7 or Hepa 1c1c7 cell lines, incubation of nuclear extracts from the MCF-7 cells treated with 100 n m MCDF with a synthetic consensus dioxin responsive element (an oligonucleotide duplex of 26 bases) gave a retarded band in a gel-retardation assay. The data suggest that the antiestrogenic effects of MCDF does not require the induction of the CYP1A1 gene expression but may involve the induction of other genes.

Regulation by Estrogen through the 5′-Flanking Region of the Transforming Growth Factor α Gene

Expression of transforming growth factor alpha (TGF alpha) mRNA and protein can be stimulated by estrogens such as 17 beta-estradiol (E2) in estrogen-responsive rodent and human breast cancer cells. To ascertain if E2 can directly regulate TGF alpha expression through the 5'-flanking region of the human TGF alpha gene, E2-responsive MCF-7 or ZR-75-1 human breast cancer cells or E2-nonresponsive MDA-MB-231 breast cancer cells were transiently transfected with a plasmid containing an 1140-base pair (bp) Sac-I fragment of the TGF alpha 5'-flanking region ligated to the chloramphenicol acetyltransferase (CAT) gene. Cells that were transfected and subsequently treated with physiological concentrations of E2 (10(-11)-10(-8) M) for 24 h exhibited a 2- to 10-fold increase in CAT activity. The E2 stimulation of CAT activity was dose-dependent with an increase first found at 10(-10) M E2. The increase in CAT activity could be detected within 24-36 h after the addition of E2. There was no significant change in CAT activity in transiently transfected MDA-MB-231 cells as mediated through the TGF alpha 5'-flanking region after E2 treatment. MCF-7 cells were also transiently transfected with different fragments of the TGF alpha 5'-flanking region ligated to the luciferase gene. In the absence of E2 treatment, no detectable luciferase activity was found.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytotoxicity of diethyldithiocarbamate in human versus rodent cell lines

Diethyldithiocarbamate (DDTC) and other dithiocarbamates are currently receiving attention as potential adjuncts to traditional chemotherapy. In vitro studies with rodent cancer cell lines have consistently shown that DDTC concentrations of 0.1-1.0 micrograms/ml are highly cytotoxic. Paradoxically, however, concentrations of 10-100 micrograms/ml have been significantly less toxic. In the present study, such a 'biphasic' pattern was reproduced when 3 rodent cell lines were exposed for 1 hour to 0.001 to 1000 micrograms DDTC/ml. In contrast, in 7 human cell lines survival decreased steadily with increasing DDTC concentration (in the same dose range) without evidence of a biphasic pattern. These data might have implications for studies in which rodent cell lines are used to model the effects of dithiocarbamates in human tissues.

Subdivision of S‐phase by analysis of nuclear 5‐bromodeoxyuridine staining patterns

After pulse labeling of mammalian cells in vivo or in vitro with 5-bromodeoxyuridine (BrdUrd), followed by immunostaining with a monoclonal antibody to DNA-incorporated BrdUrd, various intranuclear staining patterns are observed. These results are obtained in various labeling, fixation, denaturation, and staining conditions. We defined five different patterns in immunoperoxidase-stained monolayers of human and rodent cancer cells and compared mean nuclear areas as measured by computerized planimetry. Furthermore, we evaluated frequency distributions of these patterns in partly synchronized cell populations and correlated these results with flow cytometric DNA histograms. The results indicate that the observed patterns reflect the spatial and temporal organization of DNA synthesis, which seems to be characterized by at least three successive stages of replication. Evaluation of these patterns may have various applications in studies on cell cycle kinetics.