Clinical Cancer Research Joint Metastasis Research Society-AACR Conference on Metastasis Translational Cancer Medicine 2008: Cancer Clinical Trials and Personalized Medicine
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 Cell Growth & Differentiation
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 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 von Lintig, F. C.
Right arrow Articles by Boss, G. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by von Lintig, F. C.
Right arrow Articles by Boss, G. R.
Clinical Cancer Research Vol. 6, 1804-1810, May 2000
© 2000 American Association for Cancer Research

Molecular Oncology, Markers, Clinical Correlates

Ras Activation in Normal White Blood Cells and Childhood Acute Lymphoblastic Leukemia1

Friederike C. von Lintig, Ivana Huvar, Ping Law, Mitchell B. Diccianni, Alice L. Yu and Gerry R. Boss2

Departments of Medicine [F. C. v. L., I. H., P. L., G. R. B.] and Pediatrics [M. B. D., A. L. Y.] and the Cancer Center [F. C. v. L., I. H., P. L., M. B. D., A. L. Y., G. R. B.], University of California, San Diego, La Jolla, California 92093-0652


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Ras is an important cellular switch, relaying growth-promoting signals from the plasma membrane to the nucleus. In cultured cells, Ras is activated by various hematopoietic cytokines and growth factors, but the activation state of Ras in peripheral WBCs and bone marrow cells has not been studied nor has Ras activation been assessed in cells from patients with acute lymphoblastic leukemia (ALL). Using an enzyme-based method, we assessed Ras activation in peripheral WBCs, lymphocytes, and bone marrow cells from normal subjects and from children with T-cell ALL (T-ALL) and B-lineage ALL (B-ALL). In normal subjects, we found mean Ras activations of 14.3, 12.5, and 17.2% for peripheral blood WBCs, lymphocytes, and bone marrow cells, respectively. All three of these values are higher than we have found in other normal human cells, compatible with constitutive activation of Ras by cytokines and growth factors present in serum and bone marrow. In 9 of 18 children with T-ALL, Ras activation exceeded two SDs above the mean of the corresponding cells from normal subjects, whereas in none of 11 patients with B-ALL did Ras show increased activation; activating genetic mutations in ras occur in less than 10% of ALL patients. Thus, Ras is relatively activated in peripheral blood WBCs, lymphocytes, and bone marrow cells compared with other normal human cells, and Ras is activated frequently in T-ALL but not in B-ALL. Increased Ras activation in T-ALL compared with B-ALL may contribute to the more aggressive nature of the former disease.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Ras, the product of the ras proto-oncogene, is anchored at the cellular membrane and transmits proproliferative signals from cytokines and growth factors to the nucleus via MAP3 kinase and other effector proteins (1 , 2) . Ras is genetically mutated to a constitutively activated form in about one-third of all human malignancies, including about 30% of acute myelogenous leukemias (3) . Genetic ras mutations are much less frequent in ALL than in acute myelogenous leukemias occurring in only about 8% of all ALL patients and, when present, they are restricted to codons 12 and 13 of N-ras (4, 5, 6, 7) . This relatively low incidence of ras mutations in ALL has suggested that Ras plays a minor role in the pathogenesis of this leukemia (3) . However, in cell culture systems, Ras can be activated through "upstream" mechanisms, e.g., a constitutively active growth factor receptor that signals through Ras, or through "downstream" mechanisms, e.g., decreased RasGAP activity (8 , 9) . Using a highly sensitive, enzyme-based method, we have measured the activation state of Ras in several human tumors, including astrocytomas, neurosarcomas, and breast and ovarian cancers, and showed that Ras can be activated through upstream and downstream mechanisms in the absence of a genetic ras mutation (10, 11, 12) .

Because the T-l and B-cell antigen receptors as well as the interleukin (IL) 2 and 3 receptors signal, in part, through the Ras/MAP kinase pathway, it is clear that Ras is involved in regulating the growth and function of T and B cells (13, 14, 15, 16, 17, 18) . We, therefore, hypothesized that Ras may be activated in T- ALL and B-ALL by an upstream mechanism and, in the present work, we measured the activation state of Ras in children with T-ALL and B-ALL and compared the results to Ras activation in peripheral WBCs, lymphocytes, and bone marrow cells from normal subjects. We found that Ras was significantly more activated in normal blood and bone marrow cells compared with other normal human cells, consistent with stimulation of the cells by cytokines and growth factors present in serum and marrow. When compared with the normal blood and marrow cells, Ras was highly activated in half of the T-ALLs and in none of the B-ALLs. To our knowledge, this work represents the first assessment of Ras activation in normal and malignant human WBCs and lays the groundwork for novel treatment strategies of childhood T-ALL using agents that target Ras or other proteins in the Ras/MAP kinase pathway (19 , 20) . Increased Ras activation in children with T-ALL compared with B-ALL may contribute to the difference in phenotype between these two leukemias.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Harvesting and Processing of Peripheral Blood and Bone Marrow Cells.
Approximately 10 ml of peripheral blood or 1 ml of bone marrow was obtained from normal subjects and from children with B-ALL according to a procedure approved by the UCSD IRB and from children with T-ALL according to POG-approved protocols; POG samples were shipped overnight with the bone marrows diluted 1:2 in RPMI medium. To generate peripheral WBCs and nucleated bone marrow cells, red cells were lysed in 0.9% ammonium acetate according to standard procedures, and to generate peripheral blood lymphocytes, whole blood was applied to a Ficoll gradient as previously described (21) . Some of the POG samples were lymphocytes isolated on a Ficoll gradient and frozen in 10% DMSO; these samples were washed once in PBS before further processing. As discussed later in "Results," none of these isolation procedures altered the activation state of Ras.

Cell Lysis and Immunoprecipitation of Ras.
Cells were lysed by gentle shaking for 5 min at 4°C in a HEPES-based buffer containing 1% NP40, MgCl2, and protease inhibitors (10) . The lysates were centrifuged at 10,000 x g for 5 min to precipitate nuclei and other subcellular organelles, and to the supernatants were added NaCl, deoxycholate, and SDS to final concentrations of 500 mM, 0.5%, and 0.05%, respectively. The samples were split in half with one-half receiving 3 µg of the rat monoclonal anti-Ras antibody Y13–259 (experimental sample) and the other half receiving 3 µg of rat IgG (control sample); both halves received protein G agarose beads and a rabbit anti-rat IgG-Fc secondary antibody. The samples were shaken gently for 1 h at 4°C, and the immunoprecipitates were washed four times in a detergent-based buffer and two times in a Tris-based buffer. The washed immunoprecipitates were resuspended in a TrisPO4/EDTA/DTT solution and heated to 100°C for 3 min to elute GTP and GDP from the immunoprecipi-tated Ras.

We have found that a 1-h incubation of the primary antibody (Y13–259) with cell extracts is sufficient to quantitatively immunoprecipitate Ras (10) . Magnesium ion and high salt present in the incubation buffer inhibit GTP/GDP dissociation from Ras and RasGAP activity, respectively; in addition, antibody Y13–259, which is a pan-Ras antibody recognizing all three forms of Ras, i.e., H-, K- and N-Ras, is a Ras-neutralizing antibody that inhibits RasGEF and RasGAP from interacting with Ras (22) . We have shown that antibody Y13–259 is superior to antibody Y13–238 for immunoprecipitating Ras, although the latter antibody could, theoretically, precipitate Ras bound to its downstream effectors (23) . We have shown in experiments where Ras was immunoprecipitated from cells previously incubated with 32PO4 that GDP and GTP are quantitatively eluted from Ras in the TrisPO4/EDTA/DTT solution; in addition, we have shown by high-performance liquid chromatography that at neutral pH, less than 5% of GTP is destroyed when heated to 100°C for up to 5 min (10) .

Measurement of Ras Activation.
The activation state of Ras is defined as the percent of Ras molecules in the active GTP-bound state, i.e., (Ras-bound GTP/Ras-bound GTP + Ras-bound GDP) x 100 and was measured as described previously (8 , 10, 11, 12 , 24) .

Briefly, GTP that had been bound to Ras was measured in a portion of the sample by converting it to ATP using the enzyme nucleoside diphosphate kinase; ATP was measured by the luciferase/luciferin system according to the following reactions:


The assay is sensitive to 1 fmol of GTP and is linear over a two-log scale. The amount of Ras-bound GTP was determined by subtracting the control sample from the experimental sample and by using standard curves prepared with each sample set (standards prepared on separate days are within 10% of each other).

In a different portion of the sample, total guanine nucleotides bound to Ras were measured by converting GDP to GTP using pyruvate kinase and phosphoenolpyruvate and then GTP, which now represented the sum of GDP plus GTP, was measured as described above:

The amount of Ras-bound GDP was determined by subtracting the amount of Ras-bound GTP (measured in one portion of the sample) from the total amount of Ras-bound guanine nucleotides (measured in another portion of the sample). The data are expressed as femtomoles of GTP or GDP per microgram of DNA, and because of the high sensitivity and linearity of the assay, accurate data can be obtained from 5–500 x 106 cells, corresponding to approximately 0.10–10 mg DNA. As an internal control, with each sample set we measured Ras-bound GTP and GDP in HL-60 human promyelocytic leukemic cells; we have previously determined Ras activation in these cells and found it to be highly reproducible (25) .

Measurement of DNA.
DNA was measured in the nuclear fraction by a standard fluorescence method using the fluorescent dye bisbenzimidazole (26) .


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Ras Activation in Peripheral Blood WBCs, Lymphocytes, and Bone Marrow Cells from Normal Subjects.
We measured Ras activation in peripheral blood WBCs from eight subjects, in peripheral blood lymphocytes from six subjects, and in nucleated bone marrow cells from nine subjects (Table 1Citation ; Ras activation was calculated from the amounts of Ras-bound GTP and GDP that were measured, and these raw data are also shown). All of the subjects were healthy without known viral or bacterial infections, and their age range was 20 to 45 years; we did not feel it was appropriate to obtain blood and bone marrow from children for these studies. For the zero time measurements shown in Table 1Citation , the blood or bone marrow was processed immediately after collection; thus, peripheral blood WBCs or nucleated bone marrow cells were lysed within 5 min of collection, and peripheral blood lymphocytes were lysed within 30 min of collection. For the 1- and 3-h measurements, whole blood or bone marrow were kept intact at room temperature for the indicated times before processing. The overnight samples were diluted 1:2 in RPMI medium and were otherwise kept undisturbed at room temperature for 16–20 h before further processing.


View this table:
[in this window]
[in a new window]
  		Table 1

Ras activation in peripheral WBCs and bone marrow cells of normal subjects

Peripheral blood or bone marrow obtained from normal subjects was kept undisturbed at room temperature with overnight samples diluted 1:2 in RPMI medium. At the indicated times, red cells were lysed in ammonium acetate when preparing peripheral WBCs and bone marrow cells; lymphocytes were isolated on a Ficoll gradient and processed immediately or frozen as described in "Materials and Methods." Cells were extracted, Ras was immunoprecipitated, and Ras-bound GTP and GDP (Ras·GTP and ·GDP) were measured as described in "Materials and Methods"; Ras activation is (Ras·GTP/Ras·GTP + Ras·GDP). The data are the means ± SD of duplicate measurements from the indicated number of subjects.

 
We found that the activation state of Ras was 14.3 ± 3.2% (mean ± SD) in the zero time peripheral blood WBCs and that Ras activation remained stable in the 1- and 3-h and overnight samples (Table 1)Citation . This degree of Ras activation is considerably more than we have found in other normal human cells, including neuronal cells and breast and ovarian epithelial cells in which Ras activation is generally <5% [(11 , 12) and unpublished observations; in several experiments, Ras activation was measured simultaneously in peripheral WBCs and in these other normal cells]. Increased Ras activation in peripheral WBCs compared with other normal cells may occur because WBCs are continually exposed to cytokines and growth factors present in serum that signal through Ras. Consistent with this hypothesis, we found that when we changed our experimental protocol and first isolated WBCs from whole blood and then incubated the cells in the absence of serum, Ras activation levels in the cells began to decrease after about 45 min of incubation and by 90 min were reduced by about 50%. The stability of Ras activation over time in WBCs incubated in whole blood is, therefore, likely secondary to continued exposure to serum cytokines and growth factors. To determine if exposing the WBCs to 0.9% ammonium acetate during RBC removal changed the WBCs Ras activation, we modified our experimental protocol and directly added the cell lysis buffer to whole blood. Although RBCs contain little or no Ras, they do contain high amounts of ATP and, therefore, the background signal increased markedly in the absence of red cell removal; however, Ras activation under these conditions was similar to the ammonium acetate–treated cells, indicating that ammonium acetate had little or no effect on the cells’ Ras activation.

Because we were interested in determining the activation state of Ras in ALL, in which lymphocytes represent the major portion of WBCs in the peripheral blood and bone marrow, we determined the activation state of Ras in normal peripheral blood lymphocytes and found they had a degree of Ras activation similar to that of peripheral blood WBCs (Table 1)Citation . To determine if lymphocyte isolation on a Ficoll gradient affected the cell’s Ras activation, we subjected cultured human leukemic cells to the same procedure and found no change in Ras activation (data not shown). Because several of the ALL samples we analyzed were from the POG tissue bank and had been frozen for a period of time, we assessed the effect of freezing in DMSO on Ras activation in lymphocytes from five normal subjects (Table 1)Citation and in bone marrow cells from two patients with B-ALL and found no effect of the freezing on Ras activation.

The activation state of Ras in nucleated bone marrow cells was 17.2 ± 4.6% (mean ± SD) (Table 1)Citation . Although this degree of Ras activation was not statistically higher than in peripheral blood WBCs or lymphocytes (P < 0.25, two-tailed Student t test), it is consistent with marrow cells being exposed to high concentrations of cytokines and growth factors in the marrow microenvironment. In support of this idea, we found very high Ras activation (~40%) in the marrow cells of a bone marrow donor who had been treated with granulocyte colony-stimulating factor. As with peripheral blood WBCs, maintaining unseparated bone marrow at room temperature for up to 20 h before isolating the nucleated cells did not change the cells’ degree of Ras activation (Table 1)Citation . Although there was a trend toward higher amounts of Ras (i.e., the sum of Ras·GTP plus Ras·GDP) in the bone marrow cells than in the peripheral WBCs or lymphocytes (Table 1)Citation , this small difference did not reach statistical significance.

Ras Activation in Peripheral Blood WBCs and Bone Marrow Cells of Patients with T-ALL.
We assessed Ras activation in peripheral blood WBCs from five patients with T-ALL (Table 2)Citation and in nucleated bone marrow cells from 13 other T-ALL patients (Table 3)Citation . In all of the T-ALL patients whose peripheral blood WBCs were analyzed, the number of leukemic cells in the peripheral blood exceeded 90%; the percent leukemic cells in the bone marrow was more variable, but exceeded 80%. Cytogenetic studies were not available on the bone marrow samples because these are not performed as part of POG protocols. The mean age of the T-ALL patients was 11.7 years, and all but two patients were male. The peripheral blood samples were all obtained at the time of initial diagnosis, whereas 4 of the 13 bone marrow samples (patients 4, 6, 8, and 12 in Table 3Citation ) were obtained during a relapse.


View this table:
[in this window]
[in a new window]
  		Table 2

Ras activation in peripheral WBCs of patients with T-ALL

Peripheral blood was obtained from patients with T-ALL according to POG-approved protocols and the activation state of Ras was measured as described in Table 1Citation in isolated lymphocytes. The data are the mean of duplicate measurements of each sample. NA, not available.

 

View this table:
[in this window]
[in a new window]
  		Table 3

Ras activation in bone marrow cells of patients with T-ALL

Bone marrow was obtained from patients with T-ALL according to POG-approved protocols and the Ras activation state was measured in nucleated cells as described in Table 1Citation . The data are the mean of duplicate measurements of each sample. NA, not available.

 
In the T-ALL patients, Ras activation in the peripheral blood WBCs ranged from 11.1 to 37.5% (Table 2)Citation and in the bone marrow cells from 7.8% to 41.1% (Table 3)Citation . We compared these data to Ras activation in lymphocytes and bone marrow cells of normal subjects and used 2 SDs above the mean of the normal subjects’ values as the upper limits of normal (yielding upper limits of 18.3% for lymphocytes and 26.4% for bone marrow cells, Table 1Citation ). According to these values, three of the peripheral blood WBCs (from patients 3–5, Table 2Citation ) and six of the bone marrow cells from the T-ALL patients (patients 8–13, Table 3Citation ) exceeded these limits. Thus, in 9 of the 18 T-ALL patients, Ras was abnormally activated in the leukemic cells. None of the peripheral blood WBCs had been frozen, whereas four of the bone marrow samples (patients 1, 2, 4, and 5) had been frozen in DMSO; as mentioned previously, we did not find an effect of freezing on Ras activation in normal lymphocytes (Table 1)Citation , nor did we find a change in Ras activation on freezing bone marrow samples from two patients with B-ALL. There was no correlation between Ras activation levels and the peripheral WBC count of the T-ALL patients (Tables 2Citation and 3)Citation .

Ras Activation in Peripheral Blood WBCs and Bone Marrow Cells of Patients with B-ALL.
We assessed Ras activation in peripheral blood WBCs from seven patients with B-ALL and in bone marrow cells from four patients with this disease (Table 4)Citation . The age range of the patients varied from 6 months to 15 years and 8 of the 11 patients were male. Cytogenetic studies revealed that all 11 patients were negative for the Philadelphia chromosome. In none of the patients did Ras activation exceed 2 SDs above the mean of the corresponding data of the normal subjects (cf. Table 4Citation to Table 1Citation ). Three of the four peripheral blood samples (patients 4, 5, and 7, Table 4Citation ) had been frozen, whereas all of the rest of the samples were analyzed fresh. Again, as for the T-ALL samples, there was no correlation between Ras activation levels and the peripheral blood WBC count.


View this table:
[in this window]
[in a new window]
  		Table 4

Ras activation in peripheral WBCs and bone marrow cells of patients with B-ALL

Peripheral blood or bone marrow was obtained from patients with B-ALL, and the activation state of Ras was measured in nucleated cells as described in Table 1Citation . The data are the means of duplicate measurements of each sample. NA, not available.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Ras can be activated on ligand binding to several different types of receptors, including tyrosine kinase receptors, non–tyrosine kinase receptors, and G protein–coupled receptors (2 , 27) . In most cell types, Ras activation leads to increased cellular proliferation (1 , 2) ; an exception to this rule is neuronal cells, where Ras activation can lead to cellular differentiation (28) . In hematopoietic cells, Ras is activated when a variety of cytokines and growth factors bind to their respective receptors. These factors include erythropoietin, stem cell factor, colony-stimulating factor-1, granulocyte macrophage colony-stimulating factor, granulocyte-specific colony-stimulating factor, and IL-1, -2, -3, -6, and -8 (13, 14, 15, 16, 17 , 29, 30, 31, 32, 33, 34) ; a notable exception is IL-4, which does not activate Ras in hematopoietic cells (13 , 34) .

The data on Ras activation in hematopoietic cells by cytokines and growth factors has been generated in cultured cell systems using a 32PO4 radioisotope-labeling method (35) . Because this method requires relatively prolonged incubations in phosphate-free culture medium to radioactively label Ras-bound GTP and GDP, it cannot be used to assess Ras activation in WBCs immediately after collecting peripheral blood or bone marrow. The Ras binding domain of Raf-1 can be used to capture Ras, but this method assesses only GTP-bound Ras, and because it relies on Western immunoblotting, it is only semiquantitative (36) . Using a nonradioactive assay that measures absolute amounts of GTP and GDP bound to Ras, we assessed the degree of Ras activation in normal and leukemic WBCs lysed within minutes of sample collection. The Ras activation levels of 14–17% that we found in normal peripheral blood WBCs and bone marrow cells are considerably higher than the Ras activation levels of 1–5% that we have found in other normal human cells, including neuronal cells and breast and ovarian epithelial cells [(11 , 12) and unpublished data]. The reason for relatively high levels of Ras activation in hematopoietic cells is likely from continual exposure to cytokines and growth factors present in serum and bone marrow. This is consistent with our finding that Ras activation in peripheral blood WBCs decreased relatively quickly when the cells were isolated from whole blood and incubated in the absence of serum. High Ras activation in bone marrow cells likely reflects the cells’ high rates of proliferation, with Ras sending mitogenic signals via the MAP kinase pathway (2) . Because peripheral blood WBCs are nondividing cells, this suggests that in these cells Ras may transduce other messages, for example, signals necessary for cellular chemotaxis or migration; Ras can mediate cytoskeletal changes through two other small GTP-binding proteins, Rac and Rho (2) . It will be of interest to determine whether Ras becomes more activated in peripheral blood WBCs in diseases characterized by increased serum concentrations of cytokines and growth factors, for example, acute or chronic inflammation.

Ras was significantly activated in cells from 9 of the 18 patients with T-ALL (as defined as more than 2 SDs above corresponding values of normal subjects). In seven of these nine patients, Ras activation levels were extremely high, exceeding 30%. In cultured cell systems, such high levels of Ras activation are generally found only in the presence of an activating mutation in the ras gene (10 , 11 , 35 , 37) . In NIH3T3 cells an activating ras mutation, with Ras activation levels in the range of 25–30%, is sufficient to lead to cellular transformation as assessed by morphological changes, colony formation in soft agar, and tumorigenesis in nude mice (1 , 10) . Thus, it seems likely that the Ras activation of >30% observed in seven of the T-ALL samples may contribute to the malignant phenotype of the cells. Consistent with this idea, there is a significantly higher risk for hematological relapse and a trend toward a lower rate of complete remission in the small percentage of ALL patients with activating N-ras mutations (6) . Given the low rate of N-ras mutations in ALL, about 8% (4, 5, 6) , one can estimate that these mutations could account for only one or two of the T-ALL samples in this series with increased Ras activation. Because the method we use to determine Ras activation is quantitative and measures absolute amounts of Ras-bound GTP and GDP, it can be used to assess total cellular Ras (10) ; we found no difference in Ras content between the normal subjects cells and the leukemic cells, indicating there was no increase in Ras expression in the malignant cells.

When compared with B-ALL, T-ALL is generally associated with an older age at diagnosis, male preponderance, higher leukocyte counts, central nervous system involvement, a higher incidence of a mediastinal mass, and a poorer clinical outcome (38) . The biochemical and molecular causes of the more aggressive clinical manifestations of T-ALL than of B-ALL are not well understood (38) . Our data suggest that one potential cause for some of the clinical differences between these two diseases may be higher Ras activation in T-ALL compared with B-ALL.

With the advent of highly sophisticated biochemical, immunological, and molecular techniques, leukemias can be subclassified, which can provide important prognostic information (38, 39, 40) . Whether assessing Ras activation in T-ALL will provide prognostic data requires study of a much larger patient cohort, and this work is in progress.

Ras and its downstream effectors have been the subject of intense research by the pharmaceutical industry and there are now multiple Ras inhibitors available, some of which have completed Phase I and II Clinical Trials (19) . In addition to pharmacological inhibition of Ras function, antibodies against activated Ras are being tested, and most recently, viral oncotherapeutic agents have been developed that require an activated Ras for their cytotoxic effect (41) . As these various inhibitors of Ras and its effectors become clinically available, it will be of some value to know which cancers will be most appropriate to treat with these new agents. The data presented in this work provide a rational basis for treating some childhood T-ALLs with a Ras inhibitor, and the assay for assessing Ras activation could be used a priori, before initiating therapy, to determine if a particular patient is a good candidate for one of these new therapeutic approaches.


   ACKNOWLEDGMENTS
 
We thank the Pediatric Oncology Group for providing samples.


   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 in part by a Leukemia Society of America Translational Research Program Award to G. R. B. and A. L. Y. and the Cindy Matters Fund (A. L. Y. and M. B. D.); F. v. L. was supported by the Mildred Scheel-Stiftung für Krebsforschung. Back

2 To whom requests for reprints should be addressed, at Department of Medicine, Cancer Center, University of California, San Diego, La Jolla, CA 92093-0652. Phone: (619) 534-8805; Fax: (619) 534-1421; E-mail: gboss{at}ucsd.edu Back

3 The abbreviations used are: MAP, mitogen-activated protein; ALL, acute lymphoblastic leukemia; T-ALL, T-cell ALL; B-ALL, B-lineage ALL; POG, Pediatric Oncology Group. Back

Received 11/17/99; revised 2/ 4/00; accepted 2/ 7/00.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Barbacid M. Ras genes. Annu. Rev. Biochem., 56: 779-827, 1987.[CrossRef][Medline]
  2. Campbell S. L., Khosravi-Far R., Rossman K. L., Clark G. F., Der C. J. Increasing complexity of Ras signaling. Oncogene, 17: 1395-1413, 1998.[CrossRef][Medline]
  3. Bos J. L. ras-Oncogenes in human cancer: a review. Cancer Res., 49: 4682-4689, 1989.[Abstract/Free Full Text]
  4. Senn H.-P., Tran-Thang C., Wodnar-Filipowicz A., Jiricny J., Fopp M., Gratwohl A., Signer E., Weber W., Moroni C. Mutation analysis of the N-ras proto-oncogene in active and remission phase of human acute leukemias. Int. J. Cancer, 41: 59-64, 1988.[Medline]
  5. Neri A., Knowles D. M., Greco A., McCormick F., Dalla-Favera R. Analysis of RAS oncogene mutations in human lymphoid malignancies. Proc. Natl. Acad. Sci. USA, 85: 9268-9272, 1988.[Abstract/Free Full Text]
  6. Lubbert M., Mirro J., Jr., Miller C. W., Kahan J., Issac G., Kitchingman G., Mertelsmann R., Herrmann F., McCormick F., Koeffler H. P. N-Ras gene point mutations in childhood acute lymphocytic leukemia correlate with a poor prognosis. Blood, 75: 1163-1169, 1990.[Abstract/Free Full Text]
  7. Rodenhuis S., Bos J. L., Slater R. M., Behrendt H., Van’t Veer M., Smets L. A. Absence of oncogene amplifications and occasional activation of N-ras in lymphoblastic leukemia of childhood. Blood, 67: 1698-1704, 1986.[Abstract/Free Full Text]
  8. Prigent S. A., Nagane M., Lin H., Huvar I., Boss G. R., Feramisco J. R., Cavenee W. K., Su Huang H.-J. Enhanced tumorigenic behavior of glioblastoma cells expressing a truncated EGF receptor is mediated through the Ras-Shc-Grb2 pathway. J. Biol. Chem., 271: 25639-25645, 1996.[Abstract/Free Full Text]
  9. Basu T., et al Aberrant regulation of Ras proteins in malignant tumour cells from type1 neurofibromatosis patients. Nature (Lond.), 356: 713-715, 1992.[CrossRef][Medline]
  10. Scheele J. S., Rhee J. M., Boss G. R. Determination of absolute amounts of GDP and GTP bound to Ras in mammalian cells: Comparison of parental and Ras-overproducing NIH 3T3 fibroblasts. Proc. Natl. Acad. Sci. USA, 92: 1097-1100, 1995.[Abstract/Free Full Text]
  11. Guha A., Lau N., Huvar I., Gutmann D., Provias J., Pawson T., Boss G. Ras-GTP levels are elevated in human NF1 peripheral nerve tumors. Oncogene, 12: 507-513, 1996.[Medline]
  12. Guha A., Feldkamp M., Lau N., Huvar I., Boss G., Pawson T. Proliferation of human malignant astrocytomas is dependent on Ras activation. Oncogene, 15: 2755-2765, 1997.[CrossRef][Medline]
  13. Satoh T., Nakafuku M., Miyahima A., Kaziro Y. Involvement of ras protein in signal-transduction pathways from interleukin 2, interleukin 3, and granulocyte/macrophage colony-stimulating factor, but not from interleukin 4. Proc. Natl. Acad. Sci. USA, 88: 3314-3318, 1991.[Abstract/Free Full Text]
  14. Turner B., Rapp U., App H., Greene M., Dobashi K., Reed J. Interleukin 2 induces tyrosine phosphorylation and activation of p72–74 Raf-1 kinase in a T-cell line. Proc. Natl. Acad. Sci. USA, 88: 1227-1231, 1991.[Abstract/Free Full Text]
  15. Burns L. A., Karnitz L. M., Sutor S. L., Abraham R. T. Interleukin-2-induced tyrosine phosphorylation of P52shc in T lymphocytes. J. Biol. Chem., 24: 17659-17661, 1993.
  16. Franklin R. A., Tordai A., Patel H., Gardner A. M., Johnson G. L., Gelfand E. W. Ligation of the T cell receptor complex results in activation of the Ras/Raf-1/MEK/MAPK cascade in human T lymphocytes. J. Clin. Invest., 93: 2134-2140, 1994.
  17. Nel A. E., Gupta S., Lee L., Ledbetter J. A., Kanner S. B. Ligation of the T-cell antigen receptor (TCR) induces association of hSos1, ZAP-70, phospholipase C-t1, and other phosphoproteins with Grb2 and the zeta-chain of the TCR. J. Biol. Chem., 31: 18428-18436, 1995.
  18. Harwood A. E., Cambier J. C. B cell antigen receptor cross-linking triggers rapid protein kinase C independent activation of p21ras1. J. Immunol., 151: 4513-4522, 1993.[Abstract]
  19. Gibbs J. B., Oliff A., Kohl N. E. Farnesyltransferase inhibitors: Ras research yields a potential cancer therapeutic. Cell, 77: 175-178, 1994.[CrossRef][Medline]
  20. Alessi D., Cuenda A., Cohen P., Dudley D., Saltiel A. PD 098059 is a specific inhibitor of the activation of mitogen-activated protein kinase in vitro and in vivo. J. Biol. Chem., 270: 27489-27494, 1995.[Abstract/Free Full Text]
  21. Boss G. R., Thompson L. F., Spiegelberg H. L., Pichler W. J., Seegmiller J. E. Age-dependency of lymphocyte ecto-5'-nucleotidase activity. J. Immunol., 125: 679-682, 1980.[Abstract]
  22. Hattori S., Clanton D. J., Satoh T., Nakamura S., Kaziro Y., Kawakita M., Shih T. Y. Neutralizing monoclonal antibody against ras oncogene product p21 which impairs guanine nucleotide exchange. Mol. Cell. Biol., 7: 1999-2002, 1987.[Abstract/Free Full Text]
  23. Suhasini M., Li H., Lohmann S. M., Boss G. R., Pilz R. B. Cyclic-GMP-dependent protein kinase inhibits the Ras/mitogen-activated protein kinase pathway. Mol. Cell. Biol., 18: 6983-6994, 1998.[Abstract/Free Full Text]
  24. Sharma P. M., Egawa K., Huang Y., Martin J. L., Huvar I., Boss G. R., Olefsky J. M. Inhibition of phosphatidylinositol 3-kinase activity by adenovirus-mediated gene transfer and its effect on insulin action. J. Biol. Chem., 273: 18528-18537, 1998.[Abstract/Free Full Text]
  25. Pilz R. B., Huvar I., Scheele J. S., Van den Berghe G., Boss G. R. A decrease in the intracellular guanosine 5'-triphosphate concentration is necessary for granulocytic differentiation of HL-60 cells, but growth cessation and differentiation are not associated with a charge in the activation state of Ras, the transforming principle of HL-60 cells. Cell Growth Differ., 8: 53-59, 1997.[Abstract]
  26. Brunk C., Jones K., James T. Assay for nanogram quantities of DNA in cellular homogenates. Anal. Biochem., 92: 497-500, 1979.[CrossRef][Medline]
  27. Porter A. C., Vaillancourt R. R. Tyrosine kinase receptor-activated signal transduction pathways which lead to oncogenesis. Oncogene, 16: 1343-1352, 1998.
  28. Bar-Sagi D., Feramisco J. R. Microinjection of the ras oncogene protein into PC12 cells induces morphological differentiation. Cell, 42: 841-848, 1985.[CrossRef][Medline]
  29. Bashey A., Healy L., Marshall C. J. Proliferative but not nonproliferative responses to granulocyte colony-stimulating factor are associated with rapid activation of the p21ras/MAP kinase signaling pathway. Blood, 83: 949-957, 1994.[Abstract/Free Full Text]
  30. Daeipour M., Kumar G., Amaral M. C., Nel A. E. Recombinant IL-6 activates p42 and p44 mitogen-activated protein kinases in the IL-6 responsive B cell line, AF-10. J. Immunol., 150: 4743-4753, 1993.[Abstract]
  31. Okuda K., Sanghera J. S., Pelech S. L., Kanakura Y., Hallek M., Griffin J. D., Druker B. J. Granulocyte-macrophage colony-stimulating factor, interleukin-3, and steel factor induce rapid tyrosine phosphorylation of p42 and p44 MAP kinase. Blood, 79: 2880-2887, 1992.[Abstract/Free Full Text]
  32. Damen J. E., Liu L., Cutler R. L., Krystal G. Erythropoietin stimulates the tyrosine phosphorylation of Shc and its association with Grb2 and a 145-Kd tyrosine phosphorylated protein. Blood, 82: 2296-2303, 1993.[Abstract/Free Full Text]
  33. Matsuguchi T., Salgia R., Hallek M., Eder M., Druker B., Ernst T. J., Griffin J. D. Shc phosphorylation in myeloid cells is regulated by granulocyte macrophage colony-stimulating factor, Interleukin-3, and steel factor and is constitutively increased by p210BCR/ABL. J. Biol. Chem., 7: 5016-5021, 1994.
  34. Welham M. J., Duronio V., Leslie K. B., Bowtell D., Schrader J. W. Multiple hemopoietins, with the exception of Interleukin-4, induce modification of Shc and mSos1, but not their translocation. J. Biol. Chem., 269: 21165-21176, 1994.[Abstract/Free Full Text]
  35. Satoh T., Endo M., Nakafuku M., Nakamura S., Kaziro Y. Accumulation of p21rasGTP in response to stimulation with epidermal growth factor and oncogene products with tyrosine kinase activity. Proc. Natl. Acad. Sci. USA, 87: 7926-7929, 1990.[Abstract/Free Full Text]
  36. De Rooij J., Bos J. L. Minimal Ras-binding domain of Raf1 can be used as an activation-specific probe for Ras. Oncogene, 14: 623-625, 1997.[CrossRef][Medline]
  37. Hamilton M., Wolfman A. Ha-ras, and N-ras regulate MAPK activity by distinct mechanisms in vivo. Oncogene, 16: 1417-1428, 1998.[CrossRef][Medline]
  38. Pui C.-H., Behm F. G., Crist W. M. Clinical and biologic relevance of immunologic marker studies in childhood acute lymphoblastic leukemia. Blood, 82: 343-362, 1993.[Abstract/Free Full Text]
  39. Diccianni M. B., Batova A., Yu J., Vu T., Pullen J., Amylon M., Pollock B. H., Yu A. L. Shortened survival after relapse in T-cell acute lymphoblastic leukemia patients with p16/p15 deletions. Leukemia Res., 21: 549-558, 1997.[CrossRef][Medline]
  40. Oui C.-H., Evers S. G. Acute lymphoblastic leukemia. New Engl. J. Med., 339: 605-615, 1998.[Free Full Text]
  41. Coffey M. C., Strong J. E., Forsyth P. A., Lee P. W. K. Reovirus therapy of tumors with activated Ras pathway. Science (Washington DC), 282: 1332-1334, 1998.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
J. C. Chen, S. Zhuang, T. H. Nguyen, G. R. Boss, and R. B. Pilz
Oncogenic Ras Leads to Rho Activation by Activating the Mitogen-activated Protein Kinase Pathway and Decreasing Rho-GTPase-activating Protein Activity
J. Biol. Chem., January 24, 2003; 278(5): 2807 - 2818.
[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 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 von Lintig, F. C.
Right arrow Articles by Boss, G. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by von Lintig, F. C.
Right arrow Articles by Boss, G. R.

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 Cell Growth & Differentiation