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[Cancer Research 62, 887-891, February 1, 2002]
© 2002 American Association for Cancer Research

Tumor Biology

Activated Ras Regulates the Proliferation/Apoptosis Balance and Early Survival of Developing Micrometastases1

Hemanth J. Varghese, Melanie T. M. Davidson, Ian C. MacDonald, Sylvia M. Wilson, Kishore V. Nadkarni, Alan C. Groom and Ann F. Chambers2

Department of Medical Biophysics, University of Western Ontario, London, Ontario, N6A 5C1 Canada [H. J. V., M. T. M. D., I. C. M., K. V. N., A. C. G., A. F. C.]; Department of Oncology, University of Western Ontario, London, Ontario, N6A 4L6 Canada [I. C. M., A. F. C.]; and London Regional Cancer Centre, London, Ontario, N6A 4L6 Canada [S. M. W., A. F. C.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mutant, activated ras oncogenes are found in many human cancers. Experimental studies have shown that Ras enhances metastatic ability in several cell types. However, the biological mechanisms by which Ras contributes to metastasis remain poorly understood. Our goal was to determine which steps in the formation of macroscopic metastases were affected by Ras. Green fluorescent protein-transfected NIH 3T3 and T24 H-ras-transformed (PAP2) fibroblasts were injected via mesenteric vein to target mouse liver. The proportion of cells that survived at each step of the metastatic process (at 60 min to 14 days after injection) were quantified. We found that Ras did not enhance the ability of cells to extravasate from liver sinusoids or to survive as solitary undivided cells in liver tissue. Furthermore, we found that a subset of cells from both cell lines initiated growth to form micrometastases by day 3. Only micrometastases formed by ras-transformed cells, however, persisted to form macroscopic metastases by day 14, whereas most NIH 3T3 micrometastases disappeared. We investigated this difference in maintenance of developing metastases by quantifying apoptosis and proliferation within the micrometastases. PAP2 metastases had a significantly higher proportion of proliferating cells as compared with apoptosing cells, whereas NIH 3T3 metastases had low proliferation and high apoptosis levels. Whereas the ability of Ras to induce vascular endothelial growth factor has suggested one way that Ras might affect metastatic ability (through induction of angiogenesis), our study provides in vivo evidence for a direct role for Ras in maintenance of metastatic growth via a shift in proliferation/apoptosis balance to favor metastatic growth.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Metastasis, the spread of cancer from primary tumors to distant sites in the body, is the leading cause of death from this disease. A thorough understanding of the biology of the metastatic process is important for the development and optimal use of novel anticancer therapies. The ability of cells to form metastases depends on successful progression through multiple steps, from shedding of cells by a primary tumor into the bloodstream (or lymphatics) through arrest and eventual growth in distant organs. A host of molecular events are responsible for the cellular changes that govern these biological processes, and it is the net effect of the molecular events that determines the metastatic phenotype observed in experimental and clinical situations. Genomic and cDNA transfection studies have been used to clarify the regulation of these molecular events leading to the acquisition of the metastatic phenotype. In particular, transfected activated ras oncogenes have been shown to confer metastatic ability in many cell types (1 , 2) . Members of the ras oncogene family are mutated in a high proportion of human cancers (3) , and Ras pathways are activated via other mechanisms in many others (4) . Ras and Ras-activated pathways are under active study as clinical targets for anticancer therapy (5 , 6) . Thus, understanding how Ras activation can influence metastatic ability may help in the development of novel ways to prevent or treat metastasis in human cancer.

Transfection of some cell types (e.g., NIH 3T3 fibroblasts) with ras oncogenes is sufficient to confer a metastatic phenotype (7, 8, 9) . The main consequence of Ras activation is downstream alteration in the expression of many other genes, which in turn act to regulate a variety of biological processes such as cell proliferation and differentiation, apoptosis, tumor-induced immune response, vascular remodeling, and angiogenesis (10, 11, 12, 13) . Oncogenic Ras can alter these processes through a variety of molecular mechanisms, and the net effects of these alterations are phenotypic and behavioral changes in cells resulting in increased metastatic ability (as measured by end point of detectable metastatic lesions; Ref. 14 ). Despite the wealth of information about the molecular pathways and genes regulated by Ras, little is known about which specific steps in the metastatic process are Ras dependent. We have previously developed assays to quantify the ability of cells to proceed through the sequential steps in metastasis (15, 16, 17) , and we have shown that many of the rate-limiting stages in the metastatic process occur long before macroscopic metastases are visible. Koop et al. (18) showed that tumor cell survival in the circulation and extravasation into the tissue were not affected by oncogenic ras expression, using NIH 3T3 and ras-transformed NIH 3T3 (PAP2) fibroblast cell lines in the chick chorioallantoic membrane model. That study suggested that later events (i.e., survival of cells and metastases once in the tissue) were important determinants of the metastatic ability of ras-transformed PAP2 fibroblasts.

The goals of our current study were to (a) extend the analysis of the effect of Ras in metastasis to a mammalian model and (b) identify which specific later events in the metastatic process were Ras dependent. We compared the highly metastatic H-ras-transformed NIH 3T3 (PAP2) fibroblasts and nontumorigenic control NIH 3T3 fibroblasts, injected via mesenteric vein to target mouse liver, and quantified the proportion of injected cells that progressed through each stage of the metastatic process. We used IVVM3 to directly analyze the interactions between cancer cells and the liver microvasculature during cell arrest and extravasation and histopathological analysis of tissue sections at defined times during the development of metastases to characterize the biological mechanisms underlying the ability or failure to form metastases.

Using this approach, we found that the main consequence of H-ras overexpression in the PAP2 cell line was the ability to maintain the growth and survival of early micrometastases. Both NIH 3T3 and PAP2 cells formed micrometastases by 3 days, but by 14 days, the NIH 3T3 micrometastases had disappeared, whereas those from PAP2 cells had developed into macroscopic tumors. These differences were associated with changes in the proliferation:apoptosis ratio of the cells within early micrometastases. This study identified a novel, early, and direct effect of ras oncogene expression in the metastatic process: promoting the survival and growth of micrometastases at a time prior to a need for angiogenesis.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cells and Transfection.
Murine NIH 3T3 cells and PAP2 (T24-H-ras-transformed NIH 3T3 cells) cells were maintained in DMEM (Invitrogen, Burlington, Ontario, Canada) supplemented with 10% calf serum (Invitrogen). PAP2 cells were previously derived by in vivo selection of ras-transformed NIH 3T3 cells for increased metastatic ability, which was found to be accompanied by increased levels of ras expression (19 , 20) . To aid in detection of the cells in vivo, green fluorescent protein was expressed in the cells as follows. The EGFP vector (pEGFP; Clontech, Palo Alto, CA) was transfected into both cell lines using SuperFect transfection reagent and the procedure recommended by the manufacturer (Qiagen, Mississauga, Ontario, Canada). Pools of stable transfectants were selected in growth medium containing 500 µg/ml Geneticin (G418; Invitrogen).

Northern Blot Analysis.
Total cytoplasmic RNA was extracted from the cells using the TRIzol reagent (Invitrogen) according to the procedure of Chomczynski and Sacchi (21) . RNA (10 µg/lane) was electrophoresed on a 1.1% denaturing agarose gel containing 6.8% formaldehyde and capillary-transferred to GeneScreen Plus membrane (NEN Life Science Products, Inc., Boston, MA). The Northern blot was hybridized with 32P-oligolabeled VEGF cDNA (pGEM-VPF; Ref. 22 ) and T24-H-ras cDNA (pT24C3; American Type Culture Collection, Manassas, VA) probes. RNA levels were quantified by densitometry (Storm 860 PhosphorImager SI; Molecular Dynamics, Sunnyvale, CA) and calculated as levels relative to 18S rRNA (p100D9; a kind gift from Dr. D. T. Denhardt).

Metastasis End Point Assay.
Female 6–8-week-old immunodeficient SCID mice (Charles River Laboratories, Wilmington, MA) were cared for in accordance with standards of the Canadian Council on Animal Care, under a protocol approved by the University of Western Ontario Council on Animal Care. Experimental metastasis to liver was assayed as described previously (23) . Briefly, mice were anesthetized using a ketamine/xylazine mixture (1.6 mg of ketamine and 0.08 mg of xylazine per 15 g of body mass) administered by i.p. injection. To target cells to the liver, a suspension of 2 x 105 cells in 0.1 ml of DMEM supplemented with 10% calf serum was injected into the superior mesenteric vein of each mouse. At 14 (or 28) days after injection, mice were sacrificed by CO2 asphyxiation. Livers from all mice were examined for visible surface tumors and then fixed in 10% neutral buffered formalin. All livers were then examined using the cell accounting procedure described below.

Fluorescent Nanosphere Labeling.
The EGFP fluorescence was sufficiently bright to permit detection of multicellular foci and metastases using fluorescence microscopy in vivo. An additional fluorescent labeling procedure was used to ensure accurate detection and accounting of single cells because it was found in preliminary experiments that solitary cells were difficult to detect reliably in vivo using EGFP alone. Before mesenteric injection, cells were thus fluorescence labeled with Fluoresbrite carboxylated polystyrene 48-nm-diameter nanospheres (Polysciences, Warrington, PA) as described previously (24) , which provided a stable fluorescence marker in undivided cells.

Metastasis Progression.
To examine the early progression of developing metastases, groups of mice injected with NIH 3T3-EGFP and PAP2-EGFP cells were sacrificed at 60 min, 3 days, 7 days, 14 days (end point), and 28 days (NIH 3T3-EGFP only) after injection. To determine whether any NIH 3T3-EGFP-injected mice formed metastases at times later than the 14 day end point, one group of mice was allowed to continue until day 28 before sacrifice. The PAP2-EGFP-injected animals were all sacrificed by day 14 due to the high tumor burden observed by this time.

To identify the position of the injected cells with respect to the liver vasculature (i.e., intravascular or extravascular), IVVM was performed at 60 min and day 3. The procedure for IVVM of mouse liver has been described previously (17) . Briefly, mice were anesthetized with sodium pentobarbitol (Somnotol; MTC Pharmaceuticals, Cambridge, Ontario, Canada), after which the liver was exposed by an i.p. incision, and the mouse was placed on a viewing platform on the stage of an inverted epifluorescence microscope (Nikon, Diaphot TMD). Video images were viewed on a monitor and recorded on SVHS videotape. Individual cells, which were positively identified by their fluorescence against the background tissue, were assessed as being intravascular, in the process of extravasating, or extravascular. This distinction was made possible by using IVVM at high magnification. Under these conditions, the depth of focus is less than the thickness of the cells or sinusoids. Thus, by focusing up and down through the tissue, it was possible to establish whether a cell was intra- or extravascular. At the end of each experiment, the animal was sacrificed by anesthetic overdose, and the liver was removed and fixed in 10% neutral buffered formalin.

The proportion of injected cancer cells that survived in the tissue as solitary undivided cells was determined by a cell accounting technique described previously (23) . Briefly, inert 9-µm fluorescent microspheres (Bangs Laboratories, Carmel, IN) were mixed with the cells before injection at a concentration of 2 x 104 microspheres/ml (~10:1 cells:microspheres) and co-injected with the cells into the mice. The microspheres remained trapped by size restriction in the liver sinusoids and provided a reference standard for monitoring cell survival at various later time points. Formalin-fixed livers were cut to 50-µm-thick sections using a Vibratome Series 1000 sectioning system (Technical Products International, St. Louis, MO). Sections were mounted on a coverglass and viewed using an inverted fluorescence microscope (Nikon, Diaphot, TMD). Six sections from each of two lobes of the liver (12 sections in total) were analyzed to count the number of cells and microspheres in each section. To quantify the percentage of injected cells surviving in the liver, the cell:microsphere ratio in the organ at 60 min, 3 days, 7 days, 14 days, and (NIH 3T3-EGFP only) 28 days after injection was compared with the ratio in the syringe before injection. The percentage cell survival was calculated as the (cell:microsphere ratio in the liver after injection)/(cell:microsphere ratio in syringe before injection) x 100.

To assess the growth and morphology of developing micrometastases, H&E-stained histological sections of the formalin-fixed, paraffin-embedded experimental livers were analyzed by a pathologist (K. V. N.). Two sections from each of two lobes of the liver were analyzed. The total number of tumor cells present in metastases was quantified and expressed per liver section. An image of each section was digitally acquired using a flatbed scanner. These images were analyzed in Optimas 6.1 image analysis software (Optimas Corp., Bothell, WA) to quantify the total area of each liver section. The number of cells within metastases per unit liver area was calculated and used to assess tumor burden.

To determine the degree of cell proliferation and apoptosis within developing metastases, serial sections were cut adjacent to the H&E-stained sections, and immunohistochemical staining for proliferating cells using Ki-67 (monoclonal antibody; Novocastra NCL-Ki67-MM1) and apoptosing cells using the TUNEL assay were performed as described previously (23) . Sections were analyzed by a pathologist (K. V. N.), and the numbers of proliferating and apoptosing cells within each metastasis were quantified and expressed as a percentage of the total number of cells within the metastasis.

Statistical Analysis.
Statistical analysis was performed using SigmaStat for Windows v1.0 (Jandel Scientific, San Rafael, CA). All analyses were based on the number of mice. Differences between means were determined using the t test when groups passed both a normality test and an equal variance test. When this was not the case, the Mann-Whitney rank-sum test was used. A level of P < 0.05 was regarded as statistically significant.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Northern blot analysis of the parental NIH 3T3, PAP2, and the transfected NIH 3T3-EGFP and PAP2-EGFP cell lines showed that EGFP transfection did not affect H-ras expression in these cells. Thus, levels of H-Ras mRNA in the PAP2 cell lines were 5–9-fold higher than those in the NIH 3T3 control lines both before and after transfection with EGFP (Table 1)Citation . mRNA levels of VEGF, a ras-induced gene (25) , were higher in both the parental and EGFP-transfected PAP2 cell lines than in the corresponding NIH 3T3 and NIH3T3-EGFP controls (Table 1)Citation . The ability of the NIH 3T3 and ras-transformed PAP2 cells to form metastases in murine liver was assessed by experimental metastasis assay. By 2 weeks, all of the PAP2-injected mice (12 of 12 mice) had liver metastases, whereas none of the NIH 3T3-injected mice (0 of 12 mice) showed any sign of metastases (Table 1)Citation . Thus, ras-transformed fibroblasts showed enhanced metastatic ability in murine liver, extending previous results with these cell lines from chick embryo chorioallantoic membrane and mouse lung (7 , 18 , 20) . Moreover, EGFP transfection did not affect the metastatic outcome of the two cell lines (Table 1)Citation .


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  		Table 1 Gene expression and metastatic ability before and after transfection with the EGFP

H-ras and VEGF mRNA levels were assessed by Northern blot analysis in the parental cell lines and after transfection with EGFP. RNA levels were calculated from densitometry measurements and expressed relative to 18S rRNA. Metastatic ability was quantified as the fraction of mice with visible liver metastases 2 weeks after intraportal injection into mice.

 
To determine which steps in the metastatic process were affected by Ras, we used IVVM, the cell accounting technique, and histopathological analysis to quantify the survival of ras-transformed versus control cells at various times after injection to the liver. Using IVVM, we identified by direct observation the location of the injected cells with respect to the liver microvasculature. We found that at 60 min, both the control NIH 3T3-EGFP and ras-transformed PAP2-EGFP fibroblasts had arrested by size restriction within the liver sinusoids (with cessation of blood flow within these particular sinusoids), and by day 3, approximately 95% of both cell lines were fully extravascular. Thus, Ras had no effect on ability to extravasate from liver sinusoids, consistent with results obtained in the chick chorioallantoic membrane (18) .

To determine whether ras expression enhanced the ability of solitary cells to persist after extravasation, 50-µm formalin-fixed sections were analyzed, and cell survival in the tissue was calculated (Fig. 1)Citation . The rate of solitary cell loss was exponential for both cell lines, and there was no difference in this rate between control NIH3T3-EGFP and ras-transformed PAP2-EGFP fibroblasts, with >30% of cells from either cell line still present as solitary cells by day 14, and by day 28, roughly the same percentage of the NIH 3T3-EGFP cells were still present (Fig. 1)Citation . Thus, Ras had no effect on rate of loss of solitary cells, and a large population of solitary, extravasated cells remained in the liver for long periods of time.



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  		Fig. 1. Persistence and loss of solitary undivided cells. Solitary cell survival of injected cells was quantified by the cell accounting technique in 50-µm sections. At 60 min, solitary cell survival was high (>85%) for both NIH 3T3-EGFP and PAP2-EGFP cell lines. Solitary cells were lost at a near exponential rate, and this rate was similar for both cell lines up to 14 days (using an exponential curve fit, R2 = 0.953 for NIH 3T3 cells and 0.999 for PAP2 cells). {dagger}, all PAP2 cell-injected mice were sacrificed at 14 days due to high tumor burden. Bars, mean survival percentages; error bars, SE.

 
To investigate sequential steps of metastasis formation in the liver, H&E-stained histological sections were analyzed. A subset of cells from both cell lines (~1% of injected cells) had proliferated by day 3 to form micrometastases (Fig. 2, A and B)Citation , and no difference was seen in the metastatic burden between the two cell lines at this time (Fig. 3)Citation . Thus, Ras had no effect on the rate of initial proliferation to form micrometastases. However, by day 14, only a single NIH 3T3-EGFP micrometastasis was found (in 1 of 4 mice), and the remainder of the micrometastases formed by these cells had completely disappeared (Fig. 2C)Citation . Furthermore, by day 28, no micrometastases formed by NIH 3T3-EGFP cells could be found. In contrast, by day 14, all of the mice injected with PAP2-EGFP cells had formed macroscopic tumors (Fig. 2D)Citation .



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  		Fig. 2. Initiation of proliferation and persistence or loss of metastases. Histological examination (H&E staining) of paraffin-embedded liver sections from mice injected with NIH 3T3-EGFP or PAP2-EGFP cells was performed. Both cell lines initiated proliferation after extravasation to form multicellular foci in the liver tissue by day 3 (A and B). NIH 3T3-EGFP lesions were not seen at day 14, whereas all mice injected with PAP2-EGFP cells had formed macroscopic metastases by this time (C and D).

 


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  		Fig. 3. Change in tumor burden over time. The number of cells in metastases per unit liver area was quantified from H&E-stained sections. At day 3, micrometastatic lesions were visible in both experimental groups. The metastatic burden caused by the NIH 3T3-EGFP cells disappeared at an exponential rate. The metastases formed by the PAP2-EGFP cell line grew exponentially to form macroscopic metastases by 14 days. Bars, mean values; error bars, SE.

 
To identify a mechanism for the loss of NIH 3T3-EGFP micrometastases and the subsequent survival and growth of the PAP2-EGFP metastases, serial sections were assessed for proliferation (Ki-67) and apoptosis (TUNEL; Table 2Citation ). At day 3 and day 7, the number of proliferating cells within micrometastases was significantly higher in the PAP2-EGFP metastases (P < 0.001). The total number of proliferating cells decreased from day 3 to day 7 in both cell lines (P < 0.001); however, the differences between cell lines remained significant. Numbers of apoptosing cells were initially equally high in both cell lines. However, by day 7, apoptosis levels within the PAP2-EGFP metastases decreased significantly (P < 0.001). These data were used to calculate the ratio of proliferating:apoptosing cells within metastases (Fig. 4)Citation , which demonstrates differential shifts in the proliferation/apoptosis balance over time, such that growth would be favored in metastases of PAP2-EGFP, whereas loss of NIH 3T3-EGFP micrometastases would be favored.


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  		Table 2 Proliferation and apoptosis levels in metastases

Proliferation and apoptosis levels were quantified as the percentage of cells within the metastases that stained positively with Ki-67 or TUNEL, respectively, using serial histological sections.

 


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  		Fig. 4. Proliferation and apoptosis levels within metastases. The ratio of proliferation:apoptosis was calculated from data presented in Table 2Citation . Bars represent the ratio of mean percentage of proliferating cells:mean percentage of apoptosing cells.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
ras oncogenes are mutated in a large proportion of human cancers, and Ras pathways are activated by a variety of other mechanisms in many other cancers (3 , 4) . Activation of Ras-mediated signaling pathways has been associated with the metastatic phenotype (1 , 2) . Because metastasis contributes to most cancer deaths, and because therapies that target Ras and its downstream signaling pathways are under active development as anticancer agents, an understanding of the biological role of Ras in the metastatic process is clinically important. Much is known about the molecular consequences of Ras activation, and many Ras-regulated genes that could play a role in cancer cell invasion and metastasis have been identified [including members of the matrix metalloproteinase family, VEGF, osteopontin, and so forth (25, 26, 27, 28) ]. However, considerably less is known about the biological consequences of Ras activation during steps of the metastatic process.

We have developed analytical tools, using IVVM and detailed in vivo cell quantification, that permit assessment of metastatic efficiency at each step in the process (15, 16, 17) . The ability of cells to proceed through sequential steps in the metastatic process can thus be measured, and the biological effects of specific gene expression changes can be assessed in vivo. We used this approach here to determine which steps in the metastatic process are sensitive to ras expression in NIH 3T3 cells transformed by activated H-ras. We found that early steps in hematogenous, experimental metastasis to liver, including cell survival in the circulation, extravasation, and survival of individual extravasated cells, were completed with equally high efficiency by both ras-transformed (PAP2) and control NIH 3T3 cells. Ras expression, however, was found to confer a significant survival and growth advantage to small metastases. Whereas both ras-transformed and control cells initiated micrometastases from a subset of cells, only those metastases formed from the ras-transformed PAP2 cells persisted to form macroscopic metastases, whereas the micrometastases formed by control NIH 3T3 cells did not persist. This survival advantage was shown to be associated with a shift in the balance between apoptosis and proliferation within the metastases, which would preferentially favor persistence and growth of metastases that expressed activated Ras (Fig. 4)Citation . Ras can activate multiple signaling pathways, which can interact with each other as well as with signals from growth factors and integrins. These in turn can lead to proliferation, regulation of the cell cycle, and cell survival (29) , which in turn could be responsible for the ras-mediated shift in the proliferation/apoptosis balance and the metastasis survival advantage that we observed in vivo in this model.

The survival advantage conferred by Ras on metastases was detectable at a stage in the metastatic process prior to an apparent need for angiogenesis, when metastases were less than 1 mm in diameter. VEGF has been shown to be inducible by Ras, and one effect of Ras activation might therefore be to promote vascularization within primary tumors and metastases. Ras thus may affect multiple steps in the metastatic process. However, it is important to note that antiangiogenic therapies would not directly affect the Ras-mediated enhancement of survival and growth of metastases identified here, and this effect of Ras might serve to protect cancer cells in which Ras pathways were activated. Thus, apparently successful antiangiogenic treatment strategies may leave behind actively growing micrometastases, which maintain their selective growth advantage. These could lead to subsequent cancer recurrence if the antiangiogenic treatment were suspended. In addition, using IVVM, we have observed that ras-transformed and other cancer cells may preferentially seek out and grow along the outer surfaces of blood vessels (18) . Cancer cells with activated Ras pathways, if denied access to new vasculature, might continue make use of their selective survival and growth advantage and grow along preexisting vasculature, thus circumventing antiangiogenic therapeutics and developing biological "resistance" to antiangiogenic agents.

Ras and its downstream targets, either directly [e.g., farnesyl transferase inhibitors (5 , 6) ] or indirectly [e.g., anti-Her2/neu therapy (30) ], are under active development for cancer treatment. This approach offers a number of potential advantages in that the multiple biological effects of Ras could be affected. The Ras-mediated selective growth advantage demonstrated here is a clinically promising target because there is a large temporal window when this treatment strategy might be effectively used in patients. Early steps in the metastatic process, including intravasation of cancer cells from a primary tumor, arrest, extravasation, and initiation of growth of the cells to form micrometastases, have often already occurred prior to the identification of a primary tumor in many patients, and these steps are thus often not available for treatment. However, growth of metastases is a clinically available and temporally broad target. Thus, treatment strategies that target Ras and its downstream effects offer the promise of making an impact on survival of cancer patients. In the present study, we have demonstrated that a significant effect of Ras activation in cancer cells may be to confer a selective survival and growth advantage via a shift in the balance between proliferation and apoptosis within metastases.


   ACKNOWLEDGMENTS
 
We thank Nancy Kerkvliet for technical assistance with the histology and Dr. Vincent Morris for helpful discussions.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by Canadian Institutes of Health Research Grant 42511 (to A. F. C., I. C. M., and A. C. G.) and a Pre-doctoral Studentship (to H. J. V.). Back

2 To whom requests for reprints should be addressed, at the London Regional Cancer Centre, 790 Commissioners Road East, London, Ontario, N6A 4L6 Canada. Phone: (519) 685-8652; Fax: (519) 685-8646; E-mail: Ann.Chambers{at}Lrcc.on.ca Back

3 The abbreviations used are: IVVM, in vivo video microscopy; VEGF, vascular endothelial growth factor; EGFP, enhanced green fluorescent protein; TUNEL, terminal deoxynucleotidyl transferase-mediated nick end labeling. Back

Received 9/ 6/01. Accepted 11/29/01.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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  20. Chambers A. F., Denhardt G. H., Wilson S. M. ras-transformed NIH 3T3 cell lines, selected for metastatic ability in chick embryos, have increased proportions of p21-expressing cells and are metastatic in nude mice. Invasion Metastasis, 10: 225-240, 1990.[Medline]
  21. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem., 162: 156-159, 1987.[Medline]
  22. Berse B., Brown L. F., Van de Water L., Dvorak H. F., Senger D. R. Vascular permeability factor (vascular endothelial growth factor) gene is expressed differentially in normal tissues, macrophages, and tumors. Mol. Biol. Cell, 3: 211-220, 1992.[Abstract]
  23. Luzzi K. J., MacDonald I. C., Schmidt E. E., Kerkvliet N., Morris V. L., Chambers A. F., Groom A. C. Multistep nature of metastatic inefficiency: dormancy of solitary cells after successful extravasation and limited survival of early micrometastases. Am. J. Pathol., 153: 865-873, 1998.[Abstract/Free Full Text]
  24. Morris V. L., Koop S., MacDonald I. C., Schmidt E. E., Grattan M., Percy D., Chambers A. F., Groom A. C. Mammary carcinoma cell lines of high and low metastatic potential differ not in extravasation but in subsequent migration and growth. Clin. Exp. Metastasis, 12: 357-367, 1994.[Medline]
  25. Rak J., Mitsuhashi Y., Sheehan C., Tamir A., Viloria-Petit A., Filmus J., Mansour S. J., Ahn N. G., Kerbel R. S. Oncogenes and tumor angiogenesis: differential modes of vascular endothelial growth factor up-regulation in ras-transformed epithelial cells and fibroblasts. Cancer Res., 60: 490-498, 2000.[Abstract/Free Full Text]
  26. Zuber J., Tchernitsa O. I., Hinzmann B., Schmitz A. C., Grips M., Hellriegel M., Sers C., Rosenthal A., Schafer R. A genome-wide survey of RAS transformation targets. Nat. Genet., 24: 144-152, 2000.[Medline]
  27. Chambers A. F., Tuck A. B. Ras-responsive genes and tumor metastasis. Crit. Rev. Oncog., 4: 95-114, 1993.[Medline]
  28. Guo X., Zhang Y. P., Mitchell D. A., Denhardt D. T., Chambers A. F. Identification of a ras-activated enhancer in the mouse osteopontin promoter and its interaction with a putative ETS-related transcription factor whose activity correlates with the metastatic potential of the cell. Mol. Cell. Biol., 15: 476-487, 1995.[Abstract]
  29. Adjei A. A. Blocking oncogenic Ras signaling for cancer therapy. J. Natl. Cancer Inst. (Bethesda), 93: 1062-1074, 2001.[Abstract/Free Full Text]
  30. Pegram M., Slamon D. Biological rationale for HER2/neu (c-erbB2) as a target for monoclonal antibody therapy. Semin. Oncol., 27 (Suppl. 9): 13-19, 2000.



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