Skip to main content
  • AACR Publications
    • Blood Cancer Discovery
    • Cancer Discovery
    • Cancer Epidemiology, Biomarkers & Prevention
    • Cancer Immunology Research
    • Cancer Prevention Research
    • Cancer Research
    • Clinical Cancer Research
    • Molecular Cancer Research
    • Molecular Cancer Therapeutics

AACR logo

  • Register
  • Log in
  • Log out
  • My Cart
Advertisement

Main menu

  • Home
  • About
    • The Journal
    • AACR Journals
    • Subscriptions
    • Permissions and Reprints
    • Reviewing
  • Articles
    • OnlineFirst
    • Current Issue
    • Past Issues
    • Meeting Abstracts
    • Collections
      • COVID-19 & Cancer Resource Center
      • Focus on Computer Resources
      • Highly Cited Collection
      • Editors' Picks
      • "Best of" Collection
  • For Authors
    • Information for Authors
    • Author Services
    • Early Career Award
    • Best of: Author Profiles
    • Submit
  • Alerts
    • Table of Contents
    • Editors' Picks
    • OnlineFirst
    • Citations
    • Author/Keyword
    • RSS Feeds
    • My Alert Summary & Preferences
  • News
    • Cancer Discovery News
  • COVID-19
  • Webinars
  • Search More

    Advanced Search

  • AACR Publications
    • Blood Cancer Discovery
    • Cancer Discovery
    • Cancer Epidemiology, Biomarkers & Prevention
    • Cancer Immunology Research
    • Cancer Prevention Research
    • Cancer Research
    • Clinical Cancer Research
    • Molecular Cancer Research
    • Molecular Cancer Therapeutics

User menu

  • Register
  • Log in
  • Log out
  • My Cart

Search

  • Advanced search
Cancer Research
Cancer Research
  • Home
  • About
    • The Journal
    • AACR Journals
    • Subscriptions
    • Permissions and Reprints
    • Reviewing
  • Articles
    • OnlineFirst
    • Current Issue
    • Past Issues
    • Meeting Abstracts
    • Collections
      • COVID-19 & Cancer Resource Center
      • Focus on Computer Resources
      • Highly Cited Collection
      • Editors' Picks
      • "Best of" Collection
  • For Authors
    • Information for Authors
    • Author Services
    • Early Career Award
    • Best of: Author Profiles
    • Submit
  • Alerts
    • Table of Contents
    • Editors' Picks
    • OnlineFirst
    • Citations
    • Author/Keyword
    • RSS Feeds
    • My Alert Summary & Preferences
  • News
    • Cancer Discovery News
  • COVID-19
  • Webinars
  • Search More

    Advanced Search

Tumor Biology

Apoptosis: An Early Event in Metastatic Inefficiency

Christopher W. Wong, Andrea Lee, Lisa Shientag, Jong Yu, Yao Dong, Gary Kao, Abu B. Al-Mehdi, Eric J. Bernhard and Ruth J. Muschel
Christopher W. Wong
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Andrea Lee
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Lisa Shientag
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Jong Yu
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Yao Dong
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Gary Kao
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Abu B. Al-Mehdi
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Eric J. Bernhard
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Ruth J. Muschel
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
DOI:  Published January 2001
  • Article
  • Figures & Data
  • Info & Metrics
  • PDF
Loading

Abstract

Whereas large numbers of cells from a primary tumor may gain access to the circulation, few of them will give rise to metastases. The mechanism of elimination of these tumor cells, often termed“ metastatic inefficiency,” is poorly understood. In this study, we show that apoptosis in the lungs within 1–2 days of introduction of the cells is an important component of metastatic inefficiency. First, we show that death of transformed, metastatic rat embryo cells occurred via apoptosis in the lungs 24–48 h after injection into the circulation. Second, we show that Bcl-2 overexpression in these cells inhibited apoptosis in culture and also conferred resistance to apoptosis in vivo in the lungs 24–48 h after injection. This inhibition of apoptosis led to significantly more macroscopic metastases. Third, comparison between the extent of apoptosis by a poorly metastatic cell line to that by a highly metastatic cell line 24 h after injection in the lungs revealed more apoptosis by the poorly metastatic cell line. These results indicate that apoptosis, which occurs at 24–48 h after hematogenous dissemination in the lungs is an important determinant of metastatic inefficiency. Although prior work has shown an association between apoptosis in culture and metastasis in vivo, this work shows that apoptosis in vivo corresponds to decreased metastasis in vivo.

INTRODUCTION

Metastasis, the dissemination of tumor cells from a primary site to distant sites, is thought to occur by a series of steps in which tumor cells first migrate from the primary tumor, penetrate into the circulation, and eventually colonize distant sites. Evidence exists for each of these steps. Migration from the primary tumor site has been directly observed from tumors in the mammary fat pad, and intravasation from tumors in the chicken chorioallantoic membrane has been demonstrated (1 , 2) . Entrance of cells into the blood stream can be observed very rapidly after s.c. injections in experimental model systems and can also be documented in human patients (3) . Tumor cells can be found in the blood of cancer patients, although their presence often does not predict prognosis (4, 5, 6) .

Introducing tumor cells i.v. into immune-deficient experimental mice can be used as a model to duplicate the later steps of hematogenous metastasis for a wide variety of tumor cells. When lung colonies develop after i.v. injection, the cell line is regarded as having metastatic potential. In general, lung colonies form only if the cells injected are positive in spontaneous metastasis assays (i.e., if they form metastases after growth as s.c. tumors; Refs. 7 and 8 ). The injection of metastatic tumor cells into the mouse tail vein usually leads to the formation of lung, pleural, or mediastinal colonies, although colonies at other sites sometimes occur. By harvesting colonies from specific sites and selecting through several cycles of injection, it has been possible to select for cells that preferentially metastasize to these sites, such as the ovary or liver (9) . Homing to the lung can be partially attributed to the anatomy of the circulation that obliges all blood to flow through the lungs. Some have proposed that cells lodge mechanically in the pulmonary capillaries due to size constraints, but capillary occlusion cannot be sufficient in itself for growth as a metastasis because not all tumorigenic cells give rise to lung colonies (for review see Refs. 10 and 11 ). Adhesion factors have been identified that are required for some tumor cells to attach to pulmonary endothelium. For example, adhesion of B16F10 melanoma cells in the lung has been shown to depend on Lu-ECAM-1, whereas dipeptidyl peptidase IV (also known as CD26) is required for the attachment of R3230AC rat mammary and RPC-2 rat prostate carcinoma cells (12, 13, 14, 15, 16) . Esb cells derived from a murine T-cell lymphoma that metastasizes to the liver after i.v. injection instead form colonies in the muscle when β1 integrin is disrupted, but only rarely form colonies in the lung (17) . Furthermore, we have recently directly observed attachment of tumor cells to precapillary arterioles in the lung (18) .

Whereas introduction of metastatic tumor cells into the circulation results in colony formation, the majority of the injected tumor cells do not produce colonies. For example, i.v. injection of 5 × 104 cells may result in, at most, 200 colonies (19) . The failure of the majority of the cells to form colonies has been termed “metastatic inefficiency” (20) . Fidler and Nicolson (21) injected highly metastatic tumor cells labeled with 125IUDR, a radioactive thymidine analogue, into mice. By measuring radioactivity levels in various organs, they were able to determine where the injected cells migrated to after injection. These experiments indicated that the majority of cells was rapidly cleared from the blood and initially arrested in the lungs (21 , 22) . They demonstrated that by 24 h, >85% of the cells initially arrested in the lungs were lost. Two cell lines were used, one with a 8–10-fold greater ability to form pulmonary colonies than the other, yet both led to equivalent counts retained in the lungs at 2 min to 1 day (9 , 21) . These observations have been confirmed with other cell lines and with different methods of radioactive labeling by other groups (23, 24, 25, 26, 27) . The fate of these cells that fail to metastasize has not previously been demonstrated to be due to death by apoptosis.

To directly examine the fate of tumor cells within the lungs, we labeled metastatic tumor cells with GFP 3 and observed their fate in the first 1–2 days after tail-vein injection. Our observations indicated that many of the injected cells undergo apoptosis within the lungs. These results directly establish that tumor cells die in the lungs after introduction into the circulation through apoptotic processes.

MATERIALS AND METHODS

Cells and Cell Culture.

The 2.10.10 and the 3.7 rat embryo fibroblast cell lines are independent Hras and v-myc transformed metastatic lines (28) . RA3.1 is a poorly metastatic rat embryo fibroblast cell line transformed by Hras and E1A (29 , 30) . Clones overexpressing human Bcl-2 were isolated after transfection of 2.10.10 or 3.7 with pZIP-Bcl2 (kindly provided by John Reed, Burnham Institute, San Diego, CA) using lipofectamine (Life Technologies, Inc., Grand Island, NY), following the manufacturer’s directions. After selection with 200 μg/ml Geneticin (Life Technologies, Inc.), independent clones were isolated using cloning cylinders (2.10.10 Bcl-2 # 3; 2.10.10 Bcl-2 # 9). The cells were cultured at 37°C in 5% CO2 in DMEM (Life Technologies, Inc.) supplemented with penicillin-streptomycin (Life Technologies, Inc.) and 5% fetal bovine serum (Hyclone Laboratories, Inc., Logan, UT). Selection was maintained throughout.

2.10.10 and HT1080 cells expressing GFP were isolated after transfection of pEGFP-C2 (Clontech, Palo Alto, CA), 2 weeks of selection with Geneticin, followed by sorting in a flow cytometer. The 5% brightest cells were sorted and subcloned. This resulted in constitutively fluorescent clones. GFP-expressing adenovirus was a kind gift from Meenhard Herlyn (Wistar Institute, Philadelphia, PA). B16F10 cells were infected with the virus at a ratio of 20 plaque-forming units/cell, resulting in at least 90% fluorescent cells.

Radiation Treatment.

Cells (1 × 106) were plated on 10-cm dishes. The next day, they were subjected to 10 Gy of Cesium 137 irradiation (Shephard Mark I Model 68A irradiator). Twenty-four and 48 h following irradiation, cells were trypsinized, pelleted, and resuspended in 200 μl of DMEM. Cells in the medium and attached cells were pooled because apoptotic cells frequently detached. Ten microliters of propidium iodide solution [containing 3.4 mm trisodium citrate, 0.1% NP40, 4.83 mm spermine tetrahydrochlorate, 0.5 mm Tris, and 0.62 mm propidium iodide (pH 7.6)] was added to 20 μl of cell suspension, and the cells were examined under a fluorescent microscope. They were scored for apoptosis as determined by its morphology (ruffled edges, condensed chromatin and nuclear fragmentation, cell shrinkage, and formation of apoptotic bodies).

Tumorigenicity and Experimental Metastasis Assays.

Female NCR-nu/nu mice, 4–6 weeks of age, were obtained from Taconic Farms (Germantown, NY) and housed aseptically (laboratory animal facilities, University of Pennsylvania). Cells used for injection were grown to subconfluence, subjected to brief trypsin treatment, washed, and resuspended in serum-free DMEM. For tumorigenesis experiments, mice were injected bilaterally s.c. in both flanks with 5 × 105 cells in a single cell suspension in 100 μl. Tumors were measured using vernier calipers for calculation of tumor size. Six tumors were measured for each time point. For experimental metastasis assays, mice were injected with a single cell suspension of 1 × 104 cells in 100 μl into the tail vein. Animals were killed when exhibiting labored breathing or after 19 days. Lungs were fixed in 10% buffered formalin, and a dissecting microscope was used to examine lungs for evidence of metastasis.

Intravital Video Microscopy.

This assay was performed as described by Al-Mehdi et al. (18) . Briefly, mice were sacrificed by an i.p. overdose of sodium pentobarbitol at time points ranging from 30 min to 24 h after injection with cells expressing GFP. The chest was opened, and pulmonary circulation was cleared of blood by gravity flow of perfusate through a cannula inserted in the main pulmonary artery, exiting from the transected left ventricle. The perfusate was Krebs-Ringer bicarbonate solution [118.45 mm NaCl, 4.74 mm KCl, 1.17 mm MgSO4.7H2O, 1.27 mm CaCl2.2H2O, 1.18 mm KH2PO4, 24.87 mm NaHCO3 (pH 7.4), 10 mm glucose, and 5% dextran]. To visualize lung vasculature, the lungs were infused with DiI-acetylated low-density lipoprotein (Molecular Probes, Eugene, OR). The lungs were removed and examined under an inverted fluorescence microscope.

Fluorescent Microscopy.

Ten-micrometer sections were cut from mouse lung frozen in Histo Prep (Fisher Scientific, Fair Lawn, NJ). The sections were fixed in 2% paraformaldehyde and stained with 2.5 μg/ml DAPI (Sigma Chemical Co., St. Louis, MO). Green fluorescent cells were confirmed by overlay with a DAPI-stained nucleus.

Immunohistochemistry.

Ten-micrometer sections were cut from mouse lung frozen in Histo Prep (Fisher Scientific). The sections were fixed in 2% paraformaldehyde and postfixed in 2:1 ethanol:acetic acid solution. A Tris NaCl buffer[ 1 M Tris, 140 M NaCl, and 0.1% Tween 20 (pH7.6)] was used as rinsing buffer, and the sections were blocked with 5% goat serum. The primary antibody, polyclonal rabbit anti-GFP antibody, was obtained from the University of Alberta, Calgary, Canada (Ref. 31 ; 1:1500; overnight at 4°C). The alkaline phosphatase-antirabbit IgG complex was from PharMingen (San Diego, CA; 1:100; 1 h at room temperature). Levamisole (10 mg/ml; Sigma Chemical Co.) blocked any endogenous alkaline phosphatase. The chromagen used to visualize the reaction was Stable Fast Red/Naphthol Phosphate (Research Genetics, Huntsville, AL), and the sections were counterstained with aqueous hematoxylin.

TUNEL Staining.

Female nu/nu mice, 4–8 weeks of age, were given injections with a single cell suspension of 2 × 106 cells in 100 μl into the tail vein. After 24 or 48 h, the mice were sacrificed and the lungs were frozen in Histo Prep. Ten-micrometer frozen sections were made, and apoptosis was identified using the ApopTag kit (Intergen, Gaithersburg, MD) according to the manufacturer’s protocol. Briefly, sections were quenched in 2% hydrogen peroxide. The optimal dilution and incubation with the TdT enzyme was 1:54 for 1.5 h at 37°C. We used an antidigoxigenin antibody from Boehringer Manheim (Indianapolis, IN; 1:1000; 1 h at room temperature) in place of the antibody in the kit. The reaction was visualized by diaminobenzidine tetrahydrochloride (Vector Laboratories, Burlingame, CA) as the chromogen, followed by a methyl green counterstain. Spleen sections were used as a positive control with each preparation. The number of positive cells was determined by light microscopy at ×400 magnification. The slides were scanned using a digital micrometer (Microcode II; Boeckeler Instruments, Tucson, AZ) to ensure that all areas were counted only once. To determine the area of the histological sections, the slides were digitally scanned and the area of each section was calculated using Openlab software (Improvision, Coventry, United Kingdom).

Double-labeled apoptosis and anti-GFP sections were first stained with anti-GFP and visualized with Fast Red as described above, rinsed in water, and then stained with the ApopTag kit and visualized by diaminobenzidine tetrahydrochloride, as described above. The double-labeled sections were counterstained with aqueous hemotoxylin.

Immunoblotting.

Western blotting was as described by Maniatis et al. (32) . Briefly, 106 cells were plated in 10-cm tissue culture dishes. The next day, they were lysed with 200μ l of sample buffer [10% glycerol, 2% SDS, 100 mm DTT, and 50 mm Tris (pH 6.8)]. Protein samples were denatured by boiling for 5 min and run on a 12% SDS-polyacrylamide gel. After transfer onto nitrocellulose membrane (Life Technologies, Inc.), the membrane was blocked overnight in 5% milk-PBS. Bcl-2 was detected using a mouse antihuman Bcl-2 monoclonal antibody (Calbiochem, San Diego, CA) at a dilution of 1:200 and a 1:5000 dilution of secondary antibody, goat antimouse IgG horseradish peroxidase (Boehringer Mannheim). Bands were visualized using the enhanced chemiluminescence kit (Amersham Pharmacia, Piscataway, NJ).

RESULTS

Observation of GFP-labeled Tumor Cells in the Lungs after i.v. Injection.

To directly observe the fate of tumor cells in the lungs after tail-vein injection, we introduced a vector encoding enhanced GFP into two metastatic sarcoma tumor cell lines: 2.10.10, a rat embryo fibroblast transformed by Hras and v-myc, and HT 1080, a cell line derived from a human fibrosarcoma (28 , 33) . Clones 2.10.10-GFP and HT1080-GFP, which express high levels of GFP, were isolated (18) . 2.10.10-GFP cells were injected i.v. into nude mice. After 24 h the animals were sacrificed. Green fluorescent cells could readily be visualized in frozen sections of the lungs (Fig. 1A) ⇓ . Counterstaining with DAPI revealed the intact nuclei of the tumor cells surrounded by the nuclei of the cells of the lung (Fig. 1B) ⇓ . We noted that many of the green fluorescent cells appeared to have ragged edges, often with blebbing of the surface, and that the intensity of the fluorescence appeared diminished (Fig. 1C) ⇓ . The nuclei of these cells resembled apoptotic bodies (Fig. 1D) ⇓ . HT1080-GFP cells showed a similar morphology after i.v. injection (data not shown; Ref. 18 ).

Fig. 1.
  • Download figure
  • Open in new tab
  • Download powerpoint
Fig. 1.

Visualization of GFP-labeled tumor cells in the lungs. 2.10.10-GFP (2 × 106) cells were injected i.v. into nu/nu mice. The lung was harvested 24 h later, frozen in Histo Prep, sectioned, and counterstained with DAPI. A and C, DAPI staining. B and D, green fluorescence. A and B show the same section, and C and D show the same section. Arrows, tumor cells.

TUNEL staining was performed on the lung sections to determine whether the morphological changes were associated with tumor cell apoptosis. TUNEL-positive cells could readily be detected as brown after staining (Fig. 2A) ⇓ . To show that the apoptotic cells were tumor cells, we used immunohistochemistry with an antibody to GFP to identify the tumor cells. The anti-GFP antibody was visualized using alkaline phosphatase-coupled anti IgG as a secondary antibody. The detection reaction for the alkaline phosphatase gives rise to a red color. GFP-positive cells could readily be detected in the frozen sections from the lungs that had been injected with tumor cells, but not in the uninjected lungs (Fig. 2B) ⇓ . Double-staining revealed that many of the TUNEL-positive cells also contained GFP, confirming that apoptotic cells within the lung were, indeed, tumor cells (Fig. 2C) ⇓ . In similar experiments, other metastatic cell lines, including HT1080 and the murine melanoma B16F10, also gave rise to apoptotic cells within 24 h of i.v. injection into nude mice (data not shown).

Fig. 2.
  • Download figure
  • Open in new tab
  • Download powerpoint
Fig. 2.

Apoptotic 2.10.10-GFP cells in the lungs after i.v. injection. 2.10.10-GFP (2 × 106) cells were injected i.v. into nu/nu mice. After 24 h, lungs were harvested, sectioned, and stained either for apoptosis with the ApopTag kit (A), or for GFP as described in “Materials and Methods” (B), or for both (C).

Effect of Bcl-2 on Apoptosis and Metastasis.

An expression vector for Bcl-2 was introduced into 2.10.10 and another clone of transformed metastatic rat embryo cells, 3.7. Several clones that expressed Bcl-2 at markedly higher levels than the parental cells were isolated (Fig. 3) ⇓ . These cells proved to be resistant to apoptosis induced by radiation (Table 1) ⇓ . We then asked whether the number of TUNEL-positive cells in the lungs differed after the injection of the parental cells compared with injection with cells overexpressing Bcl-2. We counted the number of apoptotic cells in sections from the lungs harvested 24 or 48 h after i.v. injection of each cell type and computed the area of the sections counted (Table 2) ⇓ . Nine- to 11-fold fewer apoptotic cells were evident in the lungs of mice that received the cells overexpressing Bcl-2 at 24 or 48 h (P = 0.005). Thus, Bcl-2 protected cells from apoptosis in the lungs following i.v. injection. The number of macroscopic lung colonies formed, on average, was 5-fold greater (P ≤ 0.001) after injection of either of two different clones of the 2.10.10 cells expressing Bcl-2 relative to the parent line (Table 3) ⇓ .

Fig. 3.
  • Download figure
  • Open in new tab
  • Download powerpoint
Fig. 3.

Bcl-2 expression in transfected 2.10.10 and 3.7 clones. Lysates (60 μl) from each of the indicated cells were immunoblotted using antihuman Bcl-2.

View this table:
  • View inline
  • View popup
Table 1

Bcl-2 expression protects cells from irradiation-induced apoptosis

2.10.10 cells and those expressing Bcl-2, as shown in Fig. 3 <$REFLINK> , were irradiated with 10 Gy and harvested 24 and 48 h later.

View this table:
  • View inline
  • View popup
Table 2

Extent of apoptosis in the lungs after i.v. injection of tumor cells

Cells (2 × 106), as indicated, were injected i.v. into the tail vein of nu/nu mice. After 24 or 48 h, the mice were sacrificed and lungs were harvested, sectioned, and stained for apoptotic cells. The number of apoptotic cells on each section was counted, and the area of the section was determined. Student’s t test comparing Bcl-2 transfectants with parental cells yielded P = 0.005.

View this table:
  • View inline
  • View popup
Table 3

Bcl-2 expression enhances metastasis

2.10.10 and transfectants #3 and #9 expressing Bcl-2 (1 × 104 cells) were injected into the tail veins of nu/nu mice (five mice each in the experiment shown). The animals were sacrificed when the mice exhibited labored breathing or at day 15, and the number of lung metastasis was counted. Student’s t test comparing transfectant #3 with 2.10.10 and transfectant #9 with 2.10.10 yielded P = 0.0002 and P = 0.001, respectively. A duplicate experiment showed similar results.

Since Pietenpol et al. (34) had found that Bcl-2-overexpressing tumor cells sometimes have diminished tumorigenicity, we asked whether tumorigenicity was affected in these cells. 2.10.10 cells expressing Bcl-2 or 2.10.10 parental cells resulted in equivalent tumor growth after s.c. injection into the flanks of mice (Fig. 4) ⇓ . Thus, alteration in tumorigenicity would not seem to account for these results.

Fig. 4.
  • Download figure
  • Open in new tab
  • Download powerpoint
Fig. 4.

Bcl-2 expression does not affect growth of 2.10.10 cells as s.c. tumors. Cells (5 × 105) from 2.10.10, and two clones of 2.10.10 cells expressing Bcl-2, 2.10.10 Bcl-2 # 7 and 2.10.10 Bcl-2 # 3, were injected s.c., and tumor size was measured (see “Materials and Methods”). Student’s t tests on day 13 data yielded the following Ps: between parental and clone 3, P = 0.74; between parental and clone 7, P = 0.051; between parental and both Bcl-2 clones, P = 0.429.

Difference in Apoptosis in Vivo between Cell Lines of Different Metastatic Potential.

Since the results reported above suggest that the extent of apoptosis in vivo affects the outcome in the lung colonization assay, we determined the numbers of apoptotic cells found in the lungs after injection of two different lines of transformed rat embryo fibroblasts with different metastatic potential. Transformation of rat embryo fibroblasts by Hras with either v- or c-myc or adenovirus E1A as cooperating oncogenes results in tumorigenicity. Cells transformed by ras plus E1A, such as the cell line RA3.1, are either negative or only weakly metastatic in the lung colonization assay. In contrast, rat embryo fibroblasts transformed by Hras and myc, such as 2.10.10, are highly metastatic in this assay (29 , 33 , 35) . We compared both cell lines before injection into nu/nu mice and found that <1% were apoptotic at the time of injection. Lungs were then harvested 24 and 48 h after injection. Although both cell lines gave rise to apoptotic cells, injection of the poorly metastatic cell line resulted in significantly more apoptotic cells, especially at the 24-h time point (P = 0.003; Fig. 5 ⇓ ).

Fig. 5.
  • Download figure
  • Open in new tab
  • Download powerpoint
Fig. 5.

Comparison of the numbers of apoptotic cells in the lungs after injection of highly metastatic 2.10.10 or poorly metastatic RA3.1. 2.10.10-GFP or RA3.1-GFP (2 × 106) cells were injected i.v. into nu/nu mice. After 24 or 48 h, lungs were harvested, sectioned, and stained for apoptosis with the ApopTag kit. The number of ApopTag-positive cells was counted, and the area of the counted section was determined. At 24 h, P = 0.003; at 48 h, P = 0.132.

DISCUSSION

Our data indicate that apoptosis of tumor cells occurs in the lungs within 24 h of i.v. injection and inhibition of that apoptosis can enhance lung colonization. Tumor cells that undergo less apoptosis in the lungs were more likely to successfully establish colonies. This work suggests that apoptosis is an important component of metastatic inefficiency, at early times after initial attachment. Our recent work using intravital microscopy to examine tumor cells in the pulmonary circulation, has shown that circulating tumor cells attach to the pulmonary endothelium within 4 h in vivo or minutes in organ culture (18) . By that time, all detected tumor cells are attached and no longer blood-borne (9 , 18 , 22 , 36) . Thus, the regulation of apoptosis must be influencing metastasis of tumor cells in the early stages after attachment to the pulmonary endothelium. The attachment of tumor cells or normal cells to substratum has been shown in some instances to provide survival signals (37 , 38) . These signals may be provided by binding of integrins to extracellular matrix components (39 , 40) . Further downstream, activation of focal adhesion kinase has been shown to provide protection from apoptosis induced by lack of adhesion (41) . Apparently, the majority of the injected tumor cells, even from a metastatic cell line will not encounter the survival signals needed to remain viable and proliferate. Whether survival signals are provided through attachment to the endothelium, to extracellular matrix components on the surface of the endothelial cells, or in exposed basement membrane or through secreted molecules is yet to be established.

Survival in vivo in the circulation may be affected by mechanical factors. Although it has been proposed that cells are destroyed directly through sheer forces in the vasculature, it is also possible that mechanical stresses lead to apoptosis in susceptible cells (42 , 43) . The effect of the immune system is also unclear. In these experiments, young nude mice were used to reduce the influence of immune reactivity to tumor cells. Furthermore, there is no reason to expect that the immune system would respond within 24 h or that differential effects would be seen between the various isogenic cells used in these experiments.

The expression of genes known to affect apoptosis in culture, such as p53, Bcl-2, fas, integrin α6, or nitric oxide production affects metastasis in experimental assays. Takaoka et al. (44) observed that Bcl-2 overexpression in B16 melanoma cells strongly enhanced pulmonary metastasis, but they did not examine the effect of Bcl-2 on apoptosis in vivo. Del Bufalo et al. (45) hypothesized that Bcl-2 overexpression enhances tumorgenicity and metastatic potential of MCF7ADR cells by inducing metastasis-associated proteins. However, we found no significant differences in tumorgenicity by cells overexpressing Bcl-2 (Fig. 4) ⇓ . There are inverse correlations between the extent of induction of apoptosis in culture and metastases. The question raised by these studies is whether the signals used in culture bear any relationship to the actual signals received in animals. Nikiforov et al. (46) injected a mixture of two mouse fibroblasts, one expressing Bcl-2 and the other not expressing Bcl-2, into the tail vein of nude mice. They observed a significant enrichment of Bcl-2-expressing cells recovered 2 h after incubation in the lungs. At the same time, PCR analysis of total lung DNA revealed no change in the ratio of the injected cells. These data suggest that the depletion of non-Bcl-2-expressing cells was due to loss of viability rather than escape from lungs. However, Bcl-2 overexpression failed to alter the frequency of experimental metastasis in this case (46) . McConkey et al. (47) also reported that a nonmetastatic prostate carcinoma cell line was more susceptible to apoptosis in tissue culture, when compared with a metastatic prostate carcinoma cell line that expressed twice as much Bcl-2. Owen-Schaub et al. (48) similarly found that melanoma cells resistant to fas-mediated apoptosis were more prone to metastasize, and that fas knockout mice had higher numbers of metastasis. Xie et al. (49) have demonstrated that nitric oxide production results in apoptosis in culture and that there is an inverse correlation between apoptosis in culture and metastasis. In this study, we extend these results examining apoptosis 24–48 h in vivo after injection and show that Bcl-2 overexpression inhibits this early apoptosis. We also showed that the difference in metastatic potential between two paired cell lines correlated with the difference in apoptosis in vivo at 24–48 h after injection. Thus, the effect is in addition to the possible effects on cell proliferation, dormancy, and apoptosis that will determine the rate of nodule growth.

In other studies of metastatic inefficiency, Luzzi et al. (50) observed only 20% cell death of B16F1 after injection into the superior mesenteric vein. They attributed metastatic inefficiency to the failure of dormant solitary cells to initiate growth and failure of early micrometastases to continue growth into macroscopic tumors (50) . These results are significantly different with the results obtained after injection of radiolabeled B16F1 in that ∼70–80% of radioactivity located in the liver is lost after 24 h, suggesting extensive cell loss (21 , 22) . Cameron et al. (51) observed only 25% cell loss in the lung of mice 3 days after injection of B16F10 cells into the inferior vena cava, but 75% cell loss after 3 days. Although this correlates with the study by Luzzi et al. (50) , their observation regarding cell loss conflicts with the observations of Fidler and Nicolson (21) , also using B16F10 cells and the same mouse strain, that >90% of cells are lost within 1 day of injection (9 , 22 , 36 , 50) .

Additional data linking apoptosis to metastasis include studies examining the effect of CD44. Overexpression of a soluble CD44 fragment in a metastatic murine mammary carcinoma cell line blocked metastasis. The cells secreting this CD44 fragment underwent considerably more apoptosis in the lungs than the parental cells at 24–48 h after injection (52) .

The ability of apoptosis to modulate long-term growth of a metastatic tumor colony may be separate from the apoptotic regulation that determines early survival (24–48 h) after attachment to the pulmonary endothelia. These studies indicate that the extent of apoptosis early after pulmonary attachment correlates with the outcome in terms of formation of lung colonies.

In several cases, genes that affect apoptosis and metastasis have been found to affect the number of TUNEL staining cells when the cells were grown as tumors. Kimchi’s (53, 54, 55) laboratory isolated a gene, DAP, that bears a death domain that can interact with signals through fas or tumor necrosis factor α and whose expression affects the growth of lung carcinoma cells as tumors or as metastases. Similarly, metastatic breast carcinoma cells in which α6β1 heterodimerization is prevented grow poorly as tumors with increased apoptosis and have decreased lung colonization (56) . In these cases, whether the effect is on the ability of the cells to grow as a tumor or whether survival in the circulation is also affected has not been established. Our methods allow a distinction between survival in the circulation and later growth as a tumor. They could be applied to directly evaluate the effect of specific genes on survival in the circulation.

Acknowledgments

We are grateful for funding from the National Cancer Institute.

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 Funded by National Cancer Institute Grant CA-46830.

  • ↵2 To whom requests for reprints should be addressed, at University of Pennsylvania, 269a John Morgan Building, 3620 Hamilton Walk, Philadelphia, PA 19104. Phone: (215) 898-8401; Fax: (215) 573-4243; E-mail: muschel{at}mail.med.upenn.edu

  • ↵3 The abbreviations used are: GFP, green fluorescent protein; DAPI, 4′,6-diamidino-2-phenylindole; TUNEL, terminal deoxynucleotidyl transferase-mediated nick end labeling.

  • Received November 5, 1999.
  • Accepted November 1, 2000.
  • ©2001 American Association for Cancer Research.

References

  1. ↵
    Farina K. L., Wyckoff J. B., Rivera J., Lee H., Segall J. E., Condeelis J. S., Jones J. G. Cell motility of tumor cells visualized in living intact primary tumors using green fluorescent protein. Cancer Res., 58: 2528-2532, 1998.
    OpenUrlAbstract/FREE Full Text
  2. ↵
    Kim J., Yu W., Kovalski K., Ossowski L. Requirement for specific proteases in cancer cell intravasation as revealed by a novel semiquantitative PCR-based assay. Cell, 94: 353-362, 1998.
    OpenUrlCrossRefPubMed
  3. ↵
    Liotta L. A., Kleinerman J., Saidel G. M. Quantitative relationships of intravascular tumor cells, tumor vessels, and pulmonary metastases following tumor implantation. Cancer Res., 34: 997-1004, 1974.
    OpenUrlAbstract/FREE Full Text
  4. ↵
    Racila E., Euhus D., Weiss A. J., Rao C., McConnell J., Terstappen L. W., Uhr J. W. Detection and characterization of carcinoma cells in the blood. Proc. Natl. Acad. Sci. USA, 95: 4589-4594, 1998.
    OpenUrlAbstract/FREE Full Text
  5. ↵
    Pantel K. Detection of minimal disease in patients with solid tumors. J. Hematother., 5: 359-367, 1996.
    OpenUrlPubMed
  6. ↵
    Terstappen L. W., Rao C., Gross S., Kotelnikov V., Racilla E., Uhr J., Weiss A. Flow cytometry—principles and feasibility in transfusion medicine. Enumeration of epithelial derived tumor cells in peripheral blood. Vox Sang., 74: 269-274, 1998.
  7. ↵
    Stackpole C. W. Generation of phenotypic diversity in the B16 mouse melanoma relative to spontaneous metastasis. Cancer Res., 43: 3057-3065, 1983.
    OpenUrlAbstract/FREE Full Text
  8. ↵
    Stackpole C. W. Distinct lung-colonizing and lung-metastasizing cell populations in B16 mouse melanoma. Nature (Lond.), 289: 798-800, 1981.
    OpenUrlCrossRefPubMed
  9. ↵
    Fidler I. J., Nicolson G. L. Organ selectivity for implantation survival and growth of B16 melanoma variant tumor lines. J. Natl. Cancer Inst., 57: 1199-202, 1976.
  10. ↵
    Roos E., Dingemans K. P. Mechanisms of metastasis. Biochim. Biophys. Acta, 560: 135-166, 1979.
    OpenUrlPubMed
  11. ↵
    Morris V. L., Schmidt E. E., MacDonald I. C., Groom A. C., Chambers A. F. Sequential steps in hematogenous metastasis of cancer cells studied by in vivo videomicroscopy. Invasion Metastasis, 17: 281-296, 1997.
    OpenUrlPubMed
  12. ↵
    Cheng H. C., Abdel-Ghany M., Elble R. C., Pauli B. U. Lung endothelial dipeptidyl peptidase IV promotes adhesion and metastasis of rat breast cancer cells via tumor cell surface-associated fibronectin. J. Biol. Chem., 273: 24207-24215, 1998.
    OpenUrlAbstract/FREE Full Text
  13. ↵
    Elble R. C., Pauli B. U. Lu-ECAM-1 and DPP IV in lung metastasis. Curr. Top. Microbiol. Immunol., 213: 107-122, 1996.
  14. ↵
    Elble R. C., Widom J., Gruber A. D., Abdel-Ghany M., Levine R., Goodwin A., Cheng H. C., Pauli B. U. Cloning and characterization of lung-endothelial cell adhesion molecule-1 suggest it is an endothelial chloride channel. J. Biol. Chem., 272: 27853-27861, 1997.
    OpenUrlAbstract/FREE Full Text
  15. ↵
    Johnson R. C., Zhu D., Augustin-Voss H. G., Pauli B. U. Lung endothelial dipeptidyl peptidase IV is an adhesion molecule for lung-metastatic rat breast and prostate carcinoma cells. J Cell Biol., 121: 1423-1432, 1993.
    OpenUrlAbstract/FREE Full Text
  16. ↵
    Zhu D., Cheng C. F., Pauli B. U. Blocking of lung endothelial cell adhesion molecule-1 (Lu-ECAM-1) inhibits murine melanoma lung metastasis. J. Clin. Invest., 89: 1718-1724, 1992.
  17. ↵
    Stroeken P. J., van Rijthoven E. A., van der Valk M. A., Roos E. Targeted disruption of the β1 integrin gene in a lymphoma cell line greatly reduces metastatic capacity. Cancer Res., 58: 1569-1577, 1998.
    OpenUrlAbstract/FREE Full Text
  18. ↵
    Al-Mehdi A. B., Tozawa K., Fisher A. B., Shientag L., Lee A., Muschel R. J. Intravascular origin of metastasis from proliferation of endothelium-attached tumor cells: a new model for metastasis. Nat. Med., 6: 100-102, 2000.
    OpenUrlCrossRefPubMed
  19. ↵
    Hua J., Muschel R. J. Inhibition of matrix metalloproteinase 9 expression by a ribozyme blocks metastasis in a rat sarcoma model system. Cancer Res., 56: 5279-5284, 1996.
    OpenUrlAbstract/FREE Full Text
  20. ↵
    Weiss L. Metastatic inefficiency. Adv. Cancer Res., 54: 159-211, 1990.
    OpenUrlCrossRefPubMed
  21. ↵
    Fidler I. J., Nicolson G. L. Fate of recirculating B16 melanoma metastatic variant cells in parabiotic syngeneic recipients. J. Natl. Cancer Inst., 58: 1867-1872, 1977.
  22. ↵
    Fidler I. J. Metastasis: quantitative analysis of distribution and fate of tumor embolilabeled with 125 I-5-iodo-2′-deoxyuridine. J. Natl. Cancer Inst., 45: 773-782, 1970.
  23. ↵
    Liotta L. A., Vembu D., Saini R. K., Boone C. In vivo monitoring of the death rate of artificial murine pulmonary micrometastases. Cancer Res., 38: 1231-1236, 1978.
    OpenUrlAbstract/FREE Full Text
  24. ↵
    Aslakson C. J., Rak J. W., Miller B. E., Miller F. R. Differential influence of organ site on three subpopulations of a single mouse mammary tumor at two distinct steps in metastasis. Int. J. Cancer, 47: 466-472, 1991.
    OpenUrlPubMed
  25. ↵
    Orr F. W., Adamson I. Y., Young L. Promotion of pulmonary metastasis in mice by bleomycin-induced endothelial injury. Cancer Res., 46: 891-897, 1986.
    OpenUrlAbstract/FREE Full Text
  26. ↵
    Varani J., Lovett E. J., Elgebaly S., Lundy J., Ward P. A. In vitro and in vivo adherence of tumor cell variants correlated with tumor formation. Am. J. Pathol., 101: 345-352, 1980.
    OpenUrlPubMed
  27. ↵
    Hofer K. G., Swartzendruber D. C. Ga-citrate and I-iododeoxyuridine as markers for in vivo evaluation of tumor cell metastasis and death. J. Natl. Cancer Inst., 50: 1039-1045, 1973.
  28. ↵
    McKenna W. G., Nakahara K., Muschel R. J. Site-specific integration of H-ras in transformed rat embryo cells. Science (Washington DC), 241: 1325-1328, 1988.
    OpenUrlAbstract/FREE Full Text
  29. ↵
    Bernhard E. J., Hagner B., Wong C., Lubenski I., Muschel R. J. The effect of E1A transfection on MMP-9 expression and metastatic potential. Int. J. Cancer, 60: 718-724, 1995.
    OpenUrlCrossRefPubMed
  30. ↵
    Bernhard E. J., Gruber S. B., Muschel R. J. Direct evidence linking expression of matrix metalloproteinase 9 (92-kDa gelatinase/collagenase) to the metastatic phenotype in transformed rat embryo cells. Proc. Natl. Acad. Sci. USA, 91: 4293-4297, 1994.
    OpenUrlAbstract/FREE Full Text
  31. ↵
    Gilleard J. S., Shafi Y., Barry J. D., McGhee J. D. ELT-3: a Caenorhabditis elegans GATA factor expressed in the embryonic epidermis during morphogenesis. Dev. Biol., 208: 265-280, 1999.
    OpenUrlCrossRefPubMed
  32. ↵
    Maniatis, T., Fritsch, E. F., and Sambrook, J. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 1982.
  33. ↵
    Bernhard E. J., Muschel R. J., Hughes E. N. Mr 92,000 gelatinase release correlates with the metastatic phenotype in transformed rat embryo cells. Cancer Res., 50: 3872-3877, 1990.
    OpenUrlAbstract/FREE Full Text
  34. ↵
    Pietenpol J. A., Papadopoulos N., Markowitz S., Willson J. K., Kinzler K. W., Vogelstein B. Paradoxical inhibition of solid tumor cell growth by Bcl2. Cancer Res., 54: 3714-3717, 1994.
    OpenUrlAbstract/FREE Full Text
  35. ↵
    Pozzatti R., McCormick M., Thompson M. A., Khoury G. The E1a gene of adenovirus type 2 reduces the metastatic potential of ras-transformed rat embryo cells. Mol. Cell. Biol., 8: 2984-2988, 1988.
    OpenUrlAbstract/FREE Full Text
  36. ↵
    Fidler I. J., Nicolson G. L. Tumor cell and host properties affecting the implantation and survival of blood-borne metastatic variants of B16 melanoma. Isr J. Med. Sci., 14: 38-50, 1978.
    OpenUrlPubMed
  37. ↵
    Frisch S. M., Francis H. Disruption of epithelial cell-matrix interactions induces apoptosis. J. Cell Biol., 124: 619-626, 1994.
    OpenUrlAbstract/FREE Full Text
  38. ↵
    Boudreau N., Sympson C. J., Werb Z., Bissell M. J. Suppression of ICE and apoptosis in mammary epithelial cells by extracellular matrix. Science (Washington DC), 267: 891-893, 1995.
    OpenUrlAbstract/FREE Full Text
  39. ↵
    Boudreau N., Werb Z., Bissell M. J. Suppression of apoptosis by basement membrane requires three-dimensional tissue organization and withdrawal from the cell cycle. Proc. Natl. Acad. Sci. USA, 93: 3509-3513, 1996.
    OpenUrlAbstract/FREE Full Text
  40. ↵
    Frisch S. M., Ruoslahti E. Integrins and anoikis. Curr. Opin. Cell Biol., 9: 701-706, 1997.
    OpenUrlCrossRefPubMed
  41. ↵
    Frisch S. M., Vuori K., Ruoslahti E., Chan-Hui P. Y. Control of adhesion-dependent cell survival by focal adhesion kinase. J. Cell Biol., 134: 793-799, 1996.
    OpenUrlAbstract/FREE Full Text
  42. ↵
    Weiss L., Nannmark U., Johansson B. R., Bagge U. Lethal deformation of cancer cells in the microcirculation: a potential rate regulator of hematogenous metastasis. Int. J. Cancer, 50: 103-107, 1992.
    OpenUrlPubMed
  43. ↵
    Weiss L., Elkin G., Barbera-Guillem E. The differential resistance of B16 wild-type and F10 cells to mechanical trauma in vitro. Invasion Metastasis, 13: 92-101, 1993.
    OpenUrlPubMed
  44. ↵
    Takaoka A., Adachi M., Okuda H., Sato S., Yawata A., Hinoda Y., Takayama S., Reed J. C., Imai K. Anti-cell death activity promotes pulmonary metastasis of melanoma cells. Oncogene, 14: 2971-2977, 1997.
    OpenUrlCrossRefPubMed
  45. ↵
    Del Bufalo D., Biroccio A., Leonetti C., Zupi G. Bcl-2 overexpression enhances the metastatic potential of a human breast cancer line. FASEB J., 11: 947-953, 1997.
    OpenUrlAbstract
  46. ↵
    Nikiforov M. A., Kwek S. S., Mehta R., Artwohl J. E., Lowe S. W., Gupta T. D., Deichman G. I., Gudkov A. V. Suppression of apoptosis by Bcl-2 does not prevent p53-mediated control of experimental metastasis and anchorage dependence. Oncogene, 15: 3007-3012, 1997.
    OpenUrlCrossRefPubMed
  47. ↵
    McConkey D. J., Greene G., Pettaway C. A. Apoptosis resistance increases with metastatic potential in cells of the human LNCaP prostate carcinoma line. Cancer Res., 56: 5594-5599, 1996.
    OpenUrlAbstract/FREE Full Text
  48. ↵
    Owen-Schaub L. B., van Golen K. L., Hill L. L., Price J. E. Fas, and Fas ligand interactions suppress melanoma lung metastasis. J. Exp. Med., 188: 1717-1723, 1998.
    OpenUrlAbstract/FREE Full Text
  49. ↵
    Xie K., Huang S., Dong Z., Gutman M., Fidler I. J. Direct correlation between expression of endogenous inducible nitric oxide synthase and regression of M5076 reticulum cell sarcoma hepatic metastases in mice treated with liposomes containing lipopeptide CGP 31362. Cancer Res., 55: 3123-3131, 1995.
    OpenUrlAbstract/FREE Full Text
  50. ↵
    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.
    OpenUrlCrossRefPubMed
  51. ↵
    Cameron M. D., Schmidt E. E., Kerkvliet N., Nadkarni K. V., Morris V. L., Groom A. C., Chambers A. F., MacDonald I. C. Temporal progression of metastasis in lung: cell survival, dormancy, and location dependence of metastatic inefficiency. Cancer Res., 60: 2541-2546, 2000.
    OpenUrlAbstract/FREE Full Text
  52. ↵
    Yu Q., Toole B. P., Stamenkovic I. Induction of apoptosis of metastatic mammary carcinoma cells in vivo by disruption of tumor cell surface CD44 function. J. Exp. Med., 186: 1985-1996, 1997.
    OpenUrlAbstract/FREE Full Text
  53. ↵
    Kissil J. L., Deiss L. P., Bayewitch M., Raveh T., Khaspekov G., Kimchi A. Isolation of DAP3, a novel mediator of interferon-gamma-induced cell death. J. Biol. Chem., 270: 27932-27936, 1995.
    OpenUrlAbstract/FREE Full Text
  54. ↵
    Inbal B., Cohen O., Polak-Charcon S., Kopolovic J., Vadai E., Eisenbach L., Kimchi A. DAP kinase links the control of apoptosis to metastasis. Nature (Lond.), 390: 180-184, 1997.
    OpenUrlCrossRefPubMed
  55. ↵
    Cohen O., Inbal B., Kissil J. L., Raveh T., Berissi H., Spivak-Kroizaman T., Feinstein E., Kimchi A. DAP-kinase participates in TNF-α- and Fas-induced apoptosis and its function requires the death domain. J. Cell Biol., 146: 141-148, 1999.
    OpenUrlAbstract/FREE Full Text
  56. ↵
    Wewer U. M., Shaw L. M., Albrechtsen R., Mercurio A. M. The integrin α 6 β 1 promotes the survival of metastatic human breast carcinoma cells in mice. Am. J. Pathol., 151: 1191-1198, 1997.
    OpenUrlPubMed
PreviousNext
Back to top
Cancer Research: 61 (1)
January 2001
Volume 61, Issue 1
  • Table of Contents

Sign up for alerts

View this article with LENS

Open full page PDF
Article Alerts
Sign In to Email Alerts with your Email Address
Email Article

Thank you for sharing this Cancer Research article.

NOTE: We request your email address only to inform the recipient that it was you who recommended this article, and that it is not junk mail. We do not retain these email addresses.

Enter multiple addresses on separate lines or separate them with commas.
Apoptosis: An Early Event in Metastatic Inefficiency
(Your Name) has forwarded a page to you from Cancer Research
(Your Name) thought you would be interested in this article in Cancer Research.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Citation Tools
Apoptosis: An Early Event in Metastatic Inefficiency
Christopher W. Wong, Andrea Lee, Lisa Shientag, Jong Yu, Yao Dong, Gary Kao, Abu B. Al-Mehdi, Eric J. Bernhard and Ruth J. Muschel
Cancer Res January 1 2001 (61) (1) 333-338;

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
Share
Apoptosis: An Early Event in Metastatic Inefficiency
Christopher W. Wong, Andrea Lee, Lisa Shientag, Jong Yu, Yao Dong, Gary Kao, Abu B. Al-Mehdi, Eric J. Bernhard and Ruth J. Muschel
Cancer Res January 1 2001 (61) (1) 333-338;
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
  • Tweet Widget
  • Facebook Like
  • Google Plus One

Jump to section

  • Article
    • Abstract
    • INTRODUCTION
    • MATERIALS AND METHODS
    • RESULTS
    • DISCUSSION
    • Acknowledgments
    • Footnotes
    • References
  • Figures & Data
  • Info & Metrics
  • PDF
Advertisement

Related Articles

Cited By...

More in this TOC Section

  • Abstract 6119: RNAi rat models for drug discovery
  • Abstract 3834: Histone methyltransferase SET8 is regulated by miR-192/-215 and induces oncogene-induced senescence via p53-dependent DNA damage in human gastric carcinoma cells
  • Abstract 3788: CircHMGCS1 interacts with RNA binding protein HuR and maintains stem-like cells in gliomas
Show more Tumor Biology
  • Home
  • Alerts
  • Feedback
  • Privacy Policy
Facebook  Twitter  LinkedIn  YouTube  RSS

Articles

  • Online First
  • Current Issue
  • Past Issues
  • Meeting Abstracts

Info for

  • Authors
  • Subscribers
  • Advertisers
  • Librarians

About Cancer Research

  • About the Journal
  • Editorial Board
  • Permissions
  • Submit a Manuscript
AACR logo

Copyright © 2021 by the American Association for Cancer Research.

Cancer Research Online ISSN: 1538-7445
Cancer Research Print ISSN: 0008-5472
Journal of Cancer Research ISSN: 0099-7013
American Journal of Cancer ISSN: 0099-7374

Advertisement