Cancer Research Meeting Calendar Protein Translation and Cancer
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Fukazawa, H.
Right arrow Articles by Uehara, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Fukazawa, H.
Right arrow Articles by Uehara, Y.
[Cancer Research 60, 2104-2107, April 15, 2000]
© 2000 American Association for Cancer Research

Advances in Brief

U0126 Reverses Ki-ras-mediated Transformation by Blocking Both Mitogen-activated Protein Kinase and p70 S6 Kinase Pathways1

Hidesuke Fukazawa and Yoshimasa Uehara2

Department of Bioactive Molecules, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan


   ABSTRACT
Top
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 
U0126, a recently introduced mitogen-activated protein kinase kinase (MAPK)/extracellular signal-regulated kinase kinase inhibitor reversed morphology and inhibited anchorage-independent growth of Ki-ras-transformed rat fibroblasts. Immunoblot analyses with phosphospecific antibodies indicated that in addition to MAPK, U0126 suppressed activation of p70S6K, but not Akt, at concentrations at which it normalized the transformed phenotypes. Another MAPK/extracellular signal-regulated kinase kinase inhibitor, PD98059, showed only marginal effects on p70S6K phosphorylation and did not effectively block Ki-ras-induced transformation. However, simultaneous inhibition of the MAPK pathway and the p70S6K pathway by PD98059 in conjunction with the p70S6K inhibitor rapamycin essentially restored the normal phenotype. U0126 or the combination of PD98059 and rapamycin flattened morphology of v-src-transformed cells, but did not reverse anchorage independence, although activation of both MAPK and p70S6K was blocked. The results suggest that normalization of Ki-ras-induced transformed phenotypes by U0126 is a consequence of concurrent inhibition of the MAPK and p70S6K pathways. Intervention of other pathway(s) appears to be required to completely antagonize transformation by v-src. Simultaneous blockade of more than one signal transduction pathway by combining selective inhibitors might be effective in suppressing uncontrolled tumorigenic growth.


   Introduction
Top
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 REFERENCES
 
Much of the current effort in anticancer drug development focuses on signal transduction pathways. The strategy of precisely targeting abnormalities that propel uncontrolled growth is more rational and disease oriented than the traditional cell-killing approach. A potential difficulty in development of target therapies is assessment of their efficacy. Signal transduction inhibitors are inherently cytostatic, and simple cytotoxicity assays may not be relevant. Anchorage-independent growth is the hallmark of uncontrolled tumorigenic proliferation (1 , 2) . In theory, specific blockade of aberrant signal transduction would normalize the transformed phenotypes and thus render tumor cells anchorage dependent. The cells are expected to lose the ability to proliferate without firm attachment but still to be competent for growth on solid support. We developed a simple, objective, and quantitative method to measure anchorage-independent growth using microtiter plates coated with the antiadhesive polymer polyHEMA3 (3 , 4) . We anticipate the polyHEMA-coated plates will provide a system for evaluating inhibitors of oncogenic signal transduction.

The MAPK (extracellular signal-regulated kinase 1/2) pathway is considered to be one of the promising targets for signal transduction-based cancer chemotherapy. We examined the effects of U0126, a recently introduced MEK inhibitor (5) , on growth properties of Ki-ras- and v-src-transformed rat fibroblasts. U0126 selectively repressed anchorage-independent growth of Ki-ras transformed cells. Subsequent analyses revealed that U0126 blocked not only the MAPK pathway but also the p70S6K pathway. Experiments with PD98059 (6) and rapamycin (7) suggested that separate intervention of the MAPK pathway or the p70S6K pathway has little effect on Ki-ras-induced transformation, but simultaneous blockade of the two pathways restores the normal phenotype. The results imply significant benefits of combining inhibitors of signal transduction pathways. Synergy arising from combinations of selective pharmacological agents might extend the range of signal transduction-based cancer chemotherapy.


   Materials and Methods
Top
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 REFERENCES
 
Materials.
U0126 was from Promega (Madison, WI); PD98059 and rapamycin were from Calbiochem (San Diego, CA); and polyHEMA was from Sigma Chemical Co. (St. Louis, MO). Antibody against phosphorylated MAPK was from Promega, and other phosphospecific antibodies were from New England Biolabs (Beverly, MA). Antibodies against MAPK, p70S6K, and Akt were from Zymed Laboratories (South San Francisco, CA), Santa Cruz Biotechnology (Santa Cruz, CA), and New England Biolabs, respectively.

Cell Culture and Measurement of Anchorage-independent Growth.
Cell lines used in the experiments have been described (3) . Anchorage-independent growth was measured on polyHEMA-coated 96-well plates as described (3 , 4) . Briefly, 50 µl of polyHEMA solution (5 mg/ml in 95% ethanol) were pipetted into wells of 96-well plates and dried for 2 days with lids in place. Cells were inoculated in a volume of 135 µl at a density of 1000 cells per well for Ki-ras/NRK and 5000 per well for others. Inhibitors dissolved in 15 µl of medium were added, and the cells were cultured for 4 days. Fifteen microliters of 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide solution (5 mg/ml in PBS) were added, and the mixture was further incubated for 4 h. The resulting 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide formazan was solubilized by addition of 100 µl of SDS solution (20% in 10 mM HCl), and the absorbance was measured after 24 h at 570 nm and a reference wavelength of 690 nm using a microplate reader.

Immunoblotting Analysis.
Cells were seeded in a volume of 1 ml in 24-well plates at a density of 1 x 105 per well and cultured for 24 h. Cells were then treated with inhibitors for 24 h, washed with cold PBS, fixed for 10 min with 10% cold trichloroacetic acid, and lysed with 80 µl of 9 M urea and 2% Triton X-100 and 20 µl of 10% lithium dodecyl sulfate. Lysates were neutralized with 2 M Tris, passed through a 250-µl syringe (Hamilton, Reno, NV) to reduce viscosity and normalized for protein using a Pierce (Rockford, IL) bicinchoninic acid kit. After addition of bromphenol blue and DTT to 0.001% and 50 mM, respectively, proteins were electrophoresed through 10% SDS-PAGE and analyzed by immunoblotting using phosphospecific antibodies. Proteins were visualized with Renaissance Western blot chemiluminescence reagent (DuPont New England Nuclear, Boston, MA).


   Results
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 REFERENCES
 
U0126 Inhibits Anchorage-independent Growth and Normalizes Morphology of Ki-ras-transformed Cells.
Anchorage independence is the prime in vitro parameter of transformation (1 , 2) . We developed a microplate assay for efficient quantitation of anchorage-independent growth using plates coated with an antiadhesive polymer, polyHEMA (3 , 4) . This method can substitute for soft agar colony formation in many experimental situations and is more practical when many samples are to be tested.

We assessed the growth-inhibitory effects of MEK inhibitors on Ki-ras/NRK and v-src/NRK cells in polyHEMA-coated and normal tissue culture plastic plates. PD98059 inhibited growth of Ki-ras/NRK cells comparably in polyHEMA-coated and plastic plates (Fig. 1ACitation , middle panel). In contrast, U0126 clearly suppressed the growth of Ki-ras/NRK cells on the nonadhesive polyHEMA surface at concentrations lower than on plastic (Fig. 1ACitation , top panel). IC50 was 2 µM on polyHEMA, compared with 8 µM on plastic. Against the v-src/NRK cells, on the other hand, neither PD98059 nor U0126 showed preference for polyHEMA to tissue culture plastic (Fig. 1BCitation , top and middle panels).



View larger version (35K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 1. Effects of U0126, PD98059, and PD98059 plus rapamycin (RPM, 25 nM) on anchorage-independent growth in polyHEMA-coated (•) or anchorage-dependent growth in uncoated ({circ}) 96-well plates. A, Ki-ras/NRK cells. B, src/NRK cells. Each plot represents mean ± SD of quadruplicate wells.

 
Treatment of Ki-ras/NRK cells with U0126 caused apparent morphological reversion to a flat, normal appearance (Fig. 2ACitation ; 24-h treatment). Although not as clear, U0126 also flattened morphology of v-src/NRK cells (Fig. 2BCitation ; 48-h treatment). PD98059 induced a slight morphological change in Ki-ras/NRK cells at high concentrations (Fig. 2A)Citation , but the morphology of v-src/NRK cells did not change (Fig. 2B)Citation .



View larger version (65K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 2. Morphologies of Ki-ras/NRK (A) and v-src/NRK (B) cells treated with rapamycin (RPM, 25 nM), U0126, PD98059, or PD98059 plus rapamycin (25 nM). Concentrations of U0126 and PD98059 are as indicated.

 
U0126 Inhibits Both MAPK and p70S6K Pathways.
The above results indicate that the two MEK inhibitors U0126 and PD98059 differ in ability to block transformation by Ki-ras. Because the selectivity of PD98059 has been well tested (8) , we speculated that U0126 inhibits other pathway(s) in addition to the MAPK pathway.

To dissect the issue, we monitored the effects of the two compounds on activation of three serine/threonine kinases that are downstream targets of Ras (9) . Cell lysates from Ki-ras/NRK cells treated for 24 h were examined by immunoblotting with phosphospecific antibodies that recognize the activated form of MAPK, p70S6K or Akt. As shown in Fig. 3ACitation , both U0126 and PD98059 inhibited activation of MAPK, U0126 being more potent as reported (5) . Neither compound displayed any effect on phosphorylation of Akt. PD98059 at 25 µM only slightly inhibited activation of p70S6K. On the other hand, reduction of p70S6K phosphorylation by U0126 was much more apparent. Although U0126 did show selectivity toward MAPK over p70S6K, it clearly blocked p70S6K activation at high concentrations. Reduction of p70S6K phosphorylation was further manifested by the increased electrophoretic mobility of total p70S6K.



View larger version (57K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 3. Phosphorylation level of MAPK, p70S6K, and Akt of Ki-ras/NRK and v-src/NRK cells treated with U0126, PD98059, rapamycin (RPM), or PD98059 plus rapamycin. Cells were treated as described in "Materials and Methods" and analyzed by immunoblotting. Rapamycin was used at 25 nM. pMAPK, phosphorylated MAPK; pp70S6K, phosphorylated p70S6K; pAkt, phosphorylated Akt.

 
As shown above, anchorage-independent growth of v-src/NRK cells was not preferentially inhibited by U0126. However, U0126 also inhibited activation of MAPK and p70S6K of the v-src transformant, although p70S6K was less attenuated than in Ki-ras/NRK (Fig. 3B)Citation . Phosphotyrosine content of v-src/NRK cells did not change (data not shown).

PD98059 plus Rapamycin Blocks Transformation by Ki-ras.
The above results raised a possibility that U0126 normalizes Ki-ras-mediated transformation by blocking both MAPK and p70S6K pathways. The p70S6K pathway is selectively blocked by the immunosuppressant rapamycin (7 , 10) . Activation of p70S6K is mediated by the phosphatidylinositol kinase-related kinase mTOR/FK506-binding protein rapamycin-associated protein (11) . Rapamycin inhibits mTOR and hence p70S6K by forming a stable complex with FK506-binding protein 12, which binds to mTOR (12) . We tested whether simultaneous blockade of MAPK and p70S6K pathways by combination of PD98059 and rapamycin would result in similar consequences.

Rapamycin alone did not selectively inhibit anchorage-independent growth (data not shown) or alter morphology (Fig. 2A)Citation of Ki-ras/NRK cells. As expected, rapamycin completely blocked activation of p70S6K in Ki-ras/NRK cells without any effect on MAPK or Akt, and the combination of PD98059 and rapamycin reduced phosphorylation of both MAPK and p70S6K (Fig. 3A)Citation .

We examined the effect of PD98059 plus rapamycin on anchorage-independent growth and morphology of Ki-ras/NRK cells. At 25 nM, rapamycin by itself reduced the growth of Ki-ras/NRK cells to 57.8 ± 3.1 and 64.8 ± 4.8% of control in plastic plates and polyHEMA plates, respectively. As shown above, PD98059 did not show a marked effect on anchorage independence. However, when PD98059 was combined with 25 nM rapamycin, anchorage-independent growth on polyHEMA was noticeably inhibited over anchorage-dependent growth on plastic (Fig. 1ACitation , bottom panel). Ki-ras/NRK cells treated with PD98059 plus rapamycin appeared morphologically normal, similar to those treated with U0126 (Fig. 2A)Citation .

PD98059 plus rapamycin also inhibited MAPK and p70S6K in v-src/NRK (Fig. 3B)Citation . Cells treated with the combination were flatter than cells treated with U0126, which probably reflects the more potent inhibition of p70S6K phosphorylation. Despite the clear morphological alterations, anchorage-independent and -dependent growth was still equally inhibited (Fig. 1BCitation , bottom panel).

U0126 Is Effective against Other ras-transformed Cells.
We next examined whether U0126 could inhibit anchorage-independent growth of other ras-transformed fibroblasts. Results of two representative cell lines, Ki-ras/3Y1 and Ha-ras/3Y1, are shown in Fig. 4Citation . U0126 suppressed anchorage-independent growth (Fig. 4)Citation and flattened morphology (data not shown) of these two cell lines as well as of pMAM-Ki-ras, another Ki-ras transformed fibroblast tested (data not shown). On the contrary, PD98059 was ineffective in normalizing transformed phenotypes of Ki-ras/3Y1 or pMAM-Ki-ras (Fig. 4Citation ; data not shown). The results further support the hypothesis that normalization of Ki-ras-induced transformation requires inhibition of both MAPK and p70S6K pathways. PD98059, however, inhibited anchorage-independent growth (Fig. 4)Citation and induced morphological reversion (data not shown) of Ha-ras/3Y1 cells.



View larger version (24K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 4. Effects of U0126 and PD98059 on anchorage-independent growth in polyHEMA-coated (• and {blacksquare}) or anchorage-dependent growth in uncoated ({circ} and {square}) 96-well plates. • and {circ}, Ki-ras/3Y1 cells; {blacksquare} and {square}, Ha-ras/NRK cells.

 

   Discussion
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
REFERENCES
 
We have shown here that the MEK inhibitor U0126 normalizes morphology and inhibits anchorage-independent growth of Ki-ras-transformed rat fibroblasts. U0126 has been introduced as a potent and selective inhibitor of the MAPK pathway (5) . However, our immunoblot analyses with phosphospecific antibodies suggested that U0126 also blocks the p70S6K pathway, and the reversion of transformation appeared to coincide with concurrent inhibition of MAPK and p70S6K activation. The MEK inhibitor PD98059 did not reverse Ki-ras-induced transformation by itself but essentially restored the normal phenotype in conjunction with the p70S6K pathway inhibitor rapamycin. We conclude that simultaneous inhibition of the MAPK and p70S6K pathways is necessary and sufficient to block transformation induced by Ki-ras.

Although our results demonstrate that U0126 is more effective in blocking the MAPK pathway than the p70S6K pathway, it is obvious that data obtained from experiments that rely on U0126 as a MEK inhibitor must be cautiously interpreted. The mechanism by which U0126 inhibits p70S6K activation is not clear at present. PD98059 was less potent than U0126 in blocking the MAPK pathway but appeared to be more selective, as far as p70S6K is concerned.

Penuel and Martin (13) recently reported that simultaneous inhibition of MAPK and p70S6K pathways blocked transformation by v-src. PD98059 plus rapamycin reduced various parameters of transformation, including morphology and anchorage-independent growth in soft agar. However, their soft agar colony formation assay was not done in parallel with colony formation on a solid support. In our experiments, reversal of v-src mediated transformation was partial; i.e., inhibition of MAPK and p70S6K pathways flattened morphology to some extent but equally suppressed anchorage-dependent and -independent growth. The possibility that this is due to incomplete blockade of the two pathways remains, but we have thus far been unable to selectively reduce anchorage-independent growth with increased concentrations of the inhibitors and have obtained similar results with another v-src transformant. Intervention of other pathway(s), such as Stat3 signaling (14 , 15) , might be required to antagonize transformation by v-src completely.

In contrast, U0126 or PD98059 plus rapamycin was effective against all four ras transformants we tested (Fig. 4Citation ; data not shown). In the case of a Ha-ras transformant, inhibition of the MAPK pathway alone appeared to normalize transformation. U0126 also blocked anchorage-independent growth of several human cancer cell lines. Effects of U0126 on human cancer cell lines and cells transformed by other oncogenes will be reported elsewhere.

Activating mutations of the ras genes are among the frequently found abnormalities in human cancers (16) . MAPK is thought to be the key component in ras-mediated transformation (9) and is elevated in various human tumors (17 , 18) . Consequently, the MAPK cascade has been the focus of special attention as a target for a new generation of noncytotoxic antitumor drugs. Recently, a highly potent and selective MEK inhibitor was shown to suppress colon tumor growth in vivo without noticeable side effects, substantiating the concept of the MAPK cascade targeting as a mechanism-based cancer therapy (19) .

In Ki-ras transformed cells, however, inhibition of the MAPK pathway was not sufficient to repress disordered growth but required additional inhibition of the p70S6K pathway. Given the fact that uncontrolled growth of cancer cells involves aberrations of several mechanisms, the requirement of intervention of multiple downstream pathways in many other tumors can be envisaged. Because complete blockade of all elevated growth-promoting machineries may be harmful to normal cell activity, it would be important to identify the minimum subset of signaling pathways required to control oncogenic growth. Our results suggest that absolute inhibition of Ras function may not be necessary to remedy abnormal growth properties. Blocking of all Ras function by Ras antagonists such as farnesyltransferase inhibitors (20) might be overcorrection. Knowledge of necessary and sufficient pathways for reestablishment of regulated growth would open a way to more selective, noncytotoxic signal transduction-based chemotherapy. In cases in which blockade of a single pathway by a single agent is ineffectual, targeting of multiple, but a minimal number, of pathways by combinations of selective pharmacological agents could be a competent strategy for regulation of cancers.


   ACKNOWLEDGMENTS
 
We thank Drs. S. Mizuno and Y. Murakami for helpful discussions and Y. Zakabi for technical assistance.


   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 a grant-in-aid for Cancer Research from the Ministry of Education, Science, Sports and Culture of Japan, by the Social Institute Agency Contract Fund of the Japan Health Science Foundation, and by the Promotion of Fundamental Studies in Health Sciences of the Organization for Pharmaceutical Safety and Research of Japan. Back

2 To whom requests for reprints should be addressed. Back

3 The abbreviations used are: HEMA, 2-(hydroxyethyl methacrylate); MAPK, mitogen-activated protein kinase; MEK, MAPK/extracellular signal-regulated kinase kinase; NRK, normal rat kidney; mTOR, mammalian target of rapamycin. Back

Received 9/ 3/99. Accepted 3/ 6/00.


   REFERENCES
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 

  1. Macpherson I., Montagnier L. Agar suspension culture for the selective assay of cells transformed by polyoma virus. Virology, 23: 291-294, 1964.[Medline]
  2. Shin S., Freedman V. H., Risser R., Pollack R. Tumorigenicity of virus-transformed cells in nude mice is correlated specifically with anchorage independent growth in vitro, Proc. Natl. Acad. Sci. USA, 72: 4435-4439, 1975.[Abstract/Free Full Text]
  3. Fukazawa H., Mizuno S., Uehara Y. A microplate assay for quantitation of anchorage-independent growth of transformed cells. Anal. Biochem., 228: 83-90, 1995.[Medline]
  4. Fukazawa H., Nakano S., Mizuno S., Uehara Y. Inhibitors of anchorage-independent growth affect the growth of transformed cells on poly(2-hydroxyethyl methacrylate)-coated surfaces. Int. J. Cancer, 67: 876-882, 1996.[Medline]
  5. Favata M. F., Horiuchi K. Y., Manos E. J., Daulerio A. J., Stradley D. A., Feeser W. S., Van Dyk D. E., Pitts W. J., Earl R. A., Hobbs F., Copeland R. A., Magolda R. L., Scherle P. A., Trzaskos J. M. Identification of a novel inhibitor of mitogen-activated protein kinase kinase. J. Biol. Chem., 273: 18623-18632, 1998.[Abstract/Free Full Text]
  6. Dudley D. T., Pang L., Decker S. J., Bridges A. J., Saltiel A. R. A synthetic inhibitor of the mitogen-activated protein kinase cascade. Proc. Natl. Acad. Sci. USA, 92: 7686-7689, 1995.[Abstract/Free Full Text]
  7. Kuo C. J., Chung J., Fiorentino D. F., Flanagan W. M., Blenis J., Crabtree G. R. Rapamycin selectively inhibits interleukin-2 activation of p70 S6 kinase. Nature (Lond.), 358: 70-73, 1992.[Medline]
  8. Alessi D. R., Cuenda A., Cohen P., Dudley D. T., Saltiel A. R. PD 098059 is a specific inhibitor of the activation of mitogen-activated protein kinase kinase in vitro and in vivo. J. Biol. Chem., 270: 27489-27494, 1995.[Abstract/Free Full Text]
  9. Campbell S. L., Khosravi-Far R., Rossman K. L., Clark G. J., Der C. J. Increasing complexity of Ras signaling. Oncogene, 17: 1395-1413, 1998.[Medline]
  10. Price D. J., Grove J. R., Calvo V., Avruch J., Bierer B. E. Rapamycin-induced inhibition of the 70-kilodalton S6 protein kinase. Science (Washington DC), 257: 973-977, 1992.[Abstract/Free Full Text]
  11. Brown E. J., Beal P. A., Keith C. T., Chen J., Shin T. B., Schreiber S. L. Control of p70 s6 kinase by kinase activity of FRAP in vivo. Nature (Lond.), 377: 441-446, 1995.[Medline]
  12. Sabers C. J., Martin M. M., Brunn G. J., Williams J. M., Dumont F. J., Wiederrecht G., Abraham R. T. Isolation of a protein target of the FKBP12-rapamycin complex in mammalian cells. J. Biol. Chem., 270: 815-822, 1995.[Abstract/Free Full Text]
  13. Penuel E., Martin G. S. Transformation by v-Src: Ras-MAPK and PI3K-mTOR mediate parallel pathways. Mol. Biol. Cell, 10: 1693-1703, 1999.[Abstract/Free Full Text]
  14. Turkson J., Bowman T., Garcia R., Caldenhoven E., De Groot R. P., Jove R. Stat3 activation by Src induces specific gene regulation and is required for cell transformation. Mol. Cell. Biol., 18: 2545-2552, 1998.[Abstract/Free Full Text]
  15. Bromberg J. F., Horvath C. M., Besser D., Lathem W. W., Darnell J. E., Jr. Stat3 activation is required for cellular transformation by v-src. Mol. Cell. Biol., 18: 2553-2558, 1998.[Abstract/Free Full Text]
  16. Bos J. L. ras oncogenes in human cancer: a review. Cancer Res., 49: 4682-4689, 1989.[Abstract/Free Full Text]
  17. Hoshino R., Chatani Y., Yamori T., Tsuruo T., Oka H., Yoshida O., Shimada Y., Ari-i S., Wada H., Fujimoto J., Kohno M. Constitutive activation of the 41-/43-kDa mitogen-activated protein kinase signaling pathway in human tumors. Oncogene, 18: 813-822, 1999.[Medline]
  18. Sivaraman V. S., Wang H., Nuovo G. J., Malbon C. C. Hyperexpression of mitogen-activated protein kinase in human breast cancer. J. Clin. Invest., 99: 1478-1483, 1997.[Medline]
  19. Sebolt-Leopold J. S., Dudley D. T., Herrera R., Van Becelaere K., Wiland A., Gowan R. C., Tecle H., Barrett S. D., Bridges A., Przybranowski S., Leopold W. R., Saltiel A. R. Blockade of the MAP kinase pathway suppresses growth of colon tumors in vivo. Nat. Med., 5: 810-816, 1999.[Medline]
  20. Oliff A. Farnesyltransferase inhibitors: targeting the molecular basis of cancer. Biochim. Biophys. Acta, 1423: C19-C30, 1999.[Medline]



This article has been cited by other articles:


Home page
 J Biomol ScreenHome page
S. N. Anderson, D. L. Towne, D. J. Burns, and U. Warrior
A High-Throughput Soft Agar Assay for Identification of Anticancer Compound
J Biomol Screen, October 1, 2007; 12(7): 938 - 945.
[Abstract] [PDF]

Home page
 J. Immunol.Home page
R. Gutzmer, C. Diestel, S. Mommert, B. Kother, H. Stark, M. Wittmann, and T. Werfel
Histamine H4 Receptor Stimulation Suppresses IL-12p70 Production and Mediates Chemotaxis in Human Monocyte-Derived Dendritic Cells
J. Immunol., May 1, 2005; 174(9): 5224 - 5232.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
R. S. Sawhney, M. M. Cookson, B. Sharma, J. Hauser, and M. G. Brattain
Autocrine Transforming Growth Factor {alpha} Regulates Cell Adhesion by Multiple Signaling via Specific Phosphorylation Sites of p70S6 Kinase in Colon Cancer Cells
J. Biol. Chem., November 5, 2004; 279(45): 47379 - 47390.
[Abstract] [Full Text] [PDF]

Home page
 Molecular Cancer TherapeuticsHome page
H. Fukazawa, K. Noguchi, A. Masumi, Y. Murakami, and Y. Uehara
BimEL is an important determinant for induction of anoikis sensitivity by mitogen-activated protein/extracellular signal-regulated kinase kinase inhibitors
Mol. Cancer Ther., October 1, 2004; 3(10): 1281 - 1288.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
B. Bedogni, M. S. O'Neill, S. M. Welford, D. M. Bouley, A. J. Giaccia, N. C. Denko, and M. B. Powell
Topical Treatment with Inhibitors of the Phosphatidylinositol 3'-Kinase/Akt and Raf/Mitogen-Activated Protein Kinase Kinase/Extracellular Signal-Regulated Kinase Pathways Reduces Melanoma Development in Severe Combined Immunodeficient Mice
Cancer Res., April 1, 2004; 64(7): 2552 - 2560.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
L. Pesce, A. Comellas, and J. I. Sznajder
{beta}-Adrenergic agonists regulate Na-K-ATPase via p70S6k
Am J Physiol Lung Cell Mol Physiol, October 1, 2003; 285(4): L802 - L807.
[Abstract] [Full Text] [PDF]

Home page
 Molecular Cancer TherapeuticsHome page
H. Fukazawa, K. Noguchi, Y. Murakami, and Y. Uehara
Mitogen-activated Protein/Extracellular Signal-regulated Kinase Kinase (MEK) Inhibitors Restore Anoikis Sensitivity in Human Breast Cancer Cell Lines with a Constitutively Activated Extracellular- regulated Kinase (ERK) Pathway
Mol. Cancer Ther., March 1, 2002; 1(5): 303 - 309.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
H.-M. Koo, M. VanBrocklin, M. J. McWilliams, S. H. Leppla, N. S. Duesbery, and G. F. Vande Woude
Apoptosis and melanogenesis in human melanoma cells induced by anthrax lethal factor inactivation of mitogen-activated protein kinase kinase
PNAS, February 20, 2002; (2002) 52707699.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
M. Nomura, A. Kaji, Z. He, W.-Y. Ma, K.-i. Miyamoto, C. S. Yang, and Z. Dong
Inhibitory Mechanisms of Tea Polyphenols on the Ultraviolet B-activated Phosphatidylinositol 3-Kinase-dependent Pathway
J. Biol. Chem., November 30, 2001; 276(49): 46624 - 46631.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
P. Maher
How Protein Kinase C Activation Protects Nerve Cells from Oxidative Stress-Induced Cell Death
J. Neurosci., May 1, 2001; 21(9): 2929 - 2938.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
M. A. Morgan, O. Dolp, and C. W. M. Reuter
Cell-cycle-dependent activation of mitogen-activated protein kinase kinase (MEK-1/2) in myeloid leukemia cell lines and induction of growth inhibition and apoptosis by inhibitors of RAS signaling
Blood, March 15, 2001; 97(6): 1823 - 1834.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
B. K. Law, M. E. Waltner-Law, A. J. Entingh, A. Chytil, M. E. Aakre, P. Norgaard, and H. L. Moses
Salicylate-induced Growth Arrest Is Associated with Inhibition of p70s6k and Down-regulation of c-Myc, Cyclin D1, Cyclin A, and Proliferating Cell Nuclear Antigen
J. Biol. Chem., December 1, 2000; 275(49): 38261 - 38267.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
H.-M. Koo, M. VanBrocklin, M. J. McWilliams, S. H. Leppla, N. S. Duesbery, and G. F. V. Woude
Apoptosis and melanogenesis in human melanoma cells induced by anthrax lethal factor inactivation of mitogen-activated protein kinase kinase
PNAS, March 5, 2002; 99(5): 3052 - 3057.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
N. S. Duesbery, J. Resau, C. P. Webb, S. Koochekpour, H.-M. Koo, S. H. Leppla, and G. F. Vande Woude
Suppression of ras-mediated transformation and inhibition of tumor growth and angiogenesis by anthrax lethal factor, a proteolytic inhibitor of multiple MEK pathways
PNAS, March 27, 2001; 98(7): 4089 - 4094.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Biol. CellHome page
G. Pawlak and D. M. Helfman
Post-transcriptional Down-regulation of ROCKI/Rho-kinase through an MEK-dependent Pathway Leads to Cytoskeleton Disruption in Ras-transformed Fibroblasts
Mol. Biol. Cell, January 1, 2002; 13(1): 336 - 347.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Fukazawa, H.
Right arrow Articles by Uehara, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Fukazawa, H.
Right arrow Articles by Uehara, Y.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online