Clinical Cancer Research CTRC-AACR San Antonio Breast Cancer Symposium
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 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 Kornblau, S. M.
Right arrow Articles by May, W. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kornblau, S. M.
Right arrow Articles by May, W. S.
Clinical Cancer Research Vol. 6, 1401-1409, April 2000
© 2000 American Association for Cancer Research

Molecular Oncology, Markers, Clinical Correlates

BAX and PKC{alpha} Modulate the Prognostic Impact of BCL2 Expression in Acute Myelogenous Leukemia1

Steven M. Kornblau2, Ha T. Vu, Peter Ruvolo, Zeev Estrov, Susan O’Brien, Jorge Cortes, Hagop Kantarjian, Michael Andreeff and W. Stratford May

Section of Molecular Hematology and Therapy [S. M. K., H. T. V., M. A.] and Departments of Bioimmunotherapy [Z. E., S. O.] and Leukemia [J. C., H. K.], The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030-4095, and the Sealy Center for Oncology and Hematology, Department of Internal Medicine, The University of Texas Medical Branch at Galveston, Galveston, Texas 77550 [P. R., W. S. M.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Previously, we demonstrated that the level of BCL2 expression is prognostic in acute myelogenous leukemia (AML). High levels of BCL2 correlate with an adverse outcome when associated with favorable and intermediate prognosis cytogenetics (FIPC), whereas low levels portend an adverse outcome when associated with unfavorable cytogenetics (UC). Because BCL2 function can be modulated by dimerization with family members, like BAX, or by phosphorylation by protein kinase C {alpha} (PKC{alpha}), we hypothesize that the relative expression of these proteins in primary leukemic cells might alter the prognostic impact of BCL2 expression. We therefore measured BAX and PKC{alpha} protein levels in peripheral blood mononuclear cell lysates from 165 newly diagnosed AML patients and correlated the expression of these proteins with BCL2 expression, patient survival, and remission induction success. Expression levels of BAX and PKC{alpha} were normalized against a control cell line, K562. BAX and PKC{alpha} expression levels were heterogeneous and did not correlate with the percentage of blasts in the sample (R2 = 0.01 and <0.01). The median expression of both was similar across FAB groups but the range was greater for M4. A similar distribution of expression was observed in all cytogenetic groups, except that patients with inversion 16 demonstrated lower levels of BAX. Individually, neither PKC{alpha} nor BAX expression was prognostic of response to induction therapy or survival. A similar outcome was obtained when patients were stratified by cytogenetics into FIPC and UC groups. However, the ratio of either BCL2:BAX (B2:BX) or PKC{alpha}*B2:BX (PK*B2:BX) was highly prognostic. Patients with FIPC and a lower ratio (less than median) of either B2:BX or PK*B2:BX had a significantly higher remission induction rate (88 versus 69%, P = 0.04) and longer survival (median: 141 versus 80.5 weeks, P = 0.007) compared with those with ratios more than median. For patients with UC, values of either B2:BX or PK*B2:BX below the median had an inferior response rate to induction therapy (35 versus 78%, P = 0.0006) and inferior survival outcomes (median survival: 11 versus 53 weeks, P = 0.00002). Interestingly, FIPC and UC patients with antiapoptotic ratios (defined as B2:BX or PK*B2:BX more than median) had identical response rates and survival outcomes. In multivariate analyses, the compound variables of cytogenetics and B2:BX, or PK*B2:BX were independent predictors of survival. These results suggest that expression levels of proteins that affect the functional status of BCL2 modify the prognostic impact of BCL2 and suggest that the role of apoptosis in different cases of AML varies independently in the different cytogenetic subgroups.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The role of apoptosis in neoplastic pathogenesis and the effect of any tumor-associated apoptotic potential on response to therapy is an area of intense investigation. The signal transduction pathways regulating apoptosis are evolutionarily conserved with respect to proteins that modulate the biochemical mechanisms culminating in cell death (1, 2, 3, 4, 5) . Prominent among the regulatory proteins is the BCL2 family (4) of polypeptides comprised of both antiapoptotic (BCL2, BCL-XL, Bag1, Mcl1, and A1) and proapoptotic (BAX, Bad, Bak, Bid, and BCL-XS) members. The expression levels of these proteins are thought to determine a net apoptotic potential through protein-protein interactions, forming homo- and heterodimers (1) . One key pairing is the heterodimerization of BCL2 and its homologue BAX (6 , 7) . A popular paradigm holds that the relative proportion of proapoptotic BAX homodimers to antiapoptotic (neutralized) BCL2-BAX heterodimers dictates susceptibility to apoptotic death signals (1 , 8) . However, discrepancies between B2:BX3 ratios and sensitivity of cells to chemotherapy suggest that other factors are also involved such that chemosensitivity cannot be explained by simple quantitative differences in the expression of BCL2 or BAX (1) . In addition to functional modulation by interaction with other family members, the activity of BCL2 can be affected by posttranslational modification with phosphorylation (9, 10, 11) . One key regulatory site is serine 70, which must be phosphorylated for BCL2 to suppress apoptosis in murine growth factor-dependent cell lines (9 , 10) . Recent evidence suggests that PKC{alpha}, a classical isoform, can specifically phosphorylate BCL2 (11) . Furthermore, stable forced expression of exogenous PKC{alpha} in the human REH pre-B cell line, which expresses relatively high levels of unphosphorylated BCL2 because of a relative absence of mitochondrial PKC{alpha} (11) , induces mitochondrial localization and increased BCL2 phosphorylation in association with a >10-fold increase in resistance to drug-induced cell death (9) . In addition, PKC{alpha} also promotes protection against apoptosis via involvement in the phosphorylation and functional inactivation of BAD, a proapoptotic member of the BCL2 family (12) .4 The net apoptotic signal delivered by the BCL2 family may therefore depend not only on the relative ratios of pro- and antiapoptotic members but also on the degree of phosphorylation of BCL2 family members as well (11) .

We and others have evaluated the prognostic importance of BCL2 expression in AML (13, 14, 15, 16) .5 Recently, we reported that among patients with favorable [t(8;21), inversion 16, t(15;17)] or intermediate (diploid and insufficient metaphases) prognosis cytogenetics (FIPC), a high level of BCL2 expression correlated with an adverse prognosis, consistent with the findings of others (13, 14, 15, 16, 17) . However, an unexpected observation was that among patients with unfavorable prognosis cytogenetics (-5, -7, +8, 11q23, Ph1 , and miscellaneous changes; UC), the prognosis improved as the BCL2 level increased. Paradoxically, a high level of BCL2 expression has also been demonstrated to be a favorable prognostic factor in patients with myelodysplasia (18) , breast cancer (19) , and pediatric acute lymphocytic leukemia (20 , 21) . The state of posttranslational modification of BCL2 in these cases, although not determined, could possibly explain these apparent paradoxes. In support of a role for posttranslational modification, a recent study reported that high levels of expression of the PKC{alpha} and PKCß classical isoforms in leukemic cells from patients with childhood acute leukemias may represent a risk factor for resistance to induction-remission chemotherapy (22) . This finding raises the question of whether expression of PKC{alpha}, which can modulate the function of BCL2 by phosphorylation, might affect the prognostic impact of the level of BCL2 expression. To assess the importance of these modulators of BCL2 function on the prognostic impact of BCL2 expression in adult AML, we measured the level of protein expression of BAX and PKC{alpha} in 165 samples from newly diagnosed and previously untreated AML patients and determined any prognostic significance relative to the level of BCL2 expression.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Study Group.
Between October 1991 and July 1995, 376 patients with newly diagnosed, untreated AML were evaluated at the University of Texas M. D. Anderson Cancer Center. Peripheral blood samples were obtained from 218 of these patients prior to the initiation of therapy. Samples required for investigation in this study were acquired during routine diagnostic assessments in accordance with regulations and protocols sanctioned by the Human Subjects Committee of the University of Texas M. D. Anderson Cancer Center. Eight patients were excluded because they declined therapy. These same samples have been used previously to assess the levels of expression of retinoblastoma protein (23) , PCNA, Waf1, caspase 2, caspase 3 (24) , BCL2,5 AML2 (25) , AML1-Eto (26) , and the inversion 16 fusion product (27) . Consequently, sufficient samples to measure PKC{alpha} and BAX were only available from 165 patients. Clinical and pathological characteristics of these patients are presented in Table 1Citation . Patients that were included had better response rates and survival experiences than those patients from whom samples could not be obtained.


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

  		Table 1 Clinical characteristics of patients studied

 
Western Blotting.
Cell samples tested consist of mononuclear cells obtained by Ficoll-Hypaque (Pharmacia, Piscataway, NJ) fractionation. Western immunoblotting was performed on cell lysates derived from the Ficoll-generated mononuclear fraction of peripheral blood of 165 newly diagnosed patients with AML and 12 normal individuals as described previously (23) . Briefly, whole-cell lysates from 5 x 105 cells were electrophoresed through an 8–12% SDS-PAGE gradient gel, along with a sample of K562 and HL-60 cells (PKC{alpha}- and BAX-positive control) and another from the Y79 (American Type Cell Culture HTB 18) cell line (BCL2 positive control), a sample of peripheral blood mononuclear cells from normal individuals, and molecular weight markers. Proteins retained in the gel were electrotransferred to an Immobilon polyvinylidene difluoride membrane (Millipore, Bedford, MA) using a semidry Semi-Phor transfer apparatus (Hoefer Scientific Instruments, San Francisco, CA) at 0.8 mA/cm2 for 1.5 h. The membrane was blocked in PBS containing 0.05% Tween 20 (PBS-T) and 5% nonfat dry milk (Blotto) at room temperature for 1 h. The upper half of the membrane, containing molecular weight markers of Mr 45,000 and higher, was probed with PKC{alpha} polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) at a 1:200 dilution in PBS-T while the lower half of the membrane was probed with anti-BAX polyclonal antibody (28) at a 1:200 dilution in PBS-T, at room temperature for at least 1 h. BCL2 expression had been determined previously using these same samples in a separate experiment.5 The presence of uncleaved and cleaved PARP was assayed in a subset of patients by Western blot using an anti-PARP antibody (Pharmingen, San Diego, CA) at a 1:1000 dilution. After incubation of the membrane in the primary antibody, the membrane was washed for 15 min in PBS-T three times at room temperature and exposed to donkey antirabbit or sheep antimouse IgG conjugated to horseradish peroxidase (Amersham Life Science, Arlington Heights, IL) at a 1:2000 dilution in PBS-T for 0.5 h and then again washed for 15 min in PBS-T three times at room temperature. Subsequently, the membrane was exposed to an ECL chemiluminescence mixture (Amersham Life Science) for 1 min in accordance with the manufacturer’s recommendations and exposures (Hyperfilm-ECL film; Amersham Life Science) at intervals of 10 s to 5 min until maximum saturation was achieved. A characteristic blot is presented in Fig. 1Citation .



View larger version (44K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 1. Representative Western blot for BCL2 protein expression. Blots were probed with an anti-BAX or anti-PKC{alpha} antibody and were also probed for tubulin and BCL2 as internal controls to verify the presence of protein in the sample and to monitor for evidence of degradation. Cell lines K562, HL-60, and Y-79 served as BAX- and PKC{alpha}-positive controls; K562 is a BCL2 weak cell line, and Y-79 and HL60 are BCL2-positive controls. The last two lanes are from normal peripheral blood mononuclear cells (NL PBMC) and normal bone marrow mononuclear cells (NL-Marrow). Patients 4–6 had bone marrow (M) and blood (B) samples obtained simultaneously; all other patients were peripheral blood samples only. Loading was normalized for the same number of cells in each lane, not for the quantity of protein. We believe that normalization against other proteins from the same sample can be inaccurate because expression levels of so-called housekeeping genes are often variable.

 
Films demonstrating distinct bands with exposures in the linear range were analyzed by densitometry on a Personal Densitometer SI (Molecular Dynamics, Sunnyvale, CA). The measured levels of PKC{alpha}, BAX, and BCL2 expression were scored by densitometry and normalized against the respective control signals of K562 or Y79 to control for possible variations in antibody concentration or length of exposure. The quantitative results are expressed in terms of this standard ratio.

Statistical Analysis.
To assess the possible interactions between BAX, PKC{alpha}, and BCL2, three transformed variables were created. Because in the current paradigm (8) BAX and BCL2 are thought to be opposing forces in determination of the net apoptotic signal, a ratio of B2:BX was created. In contrast, because more PKC{alpha} might result in more phosphorylated and hence more active BCL2, which may offer enhanced protection against apoptosis, a multiplicative variable was created, PK*B2. Finally an interactive variable for all three considerations was generated, where B2:BX was multiplied by PKC{alpha} (PK*B2:BX). Because these variables have antiapoptotic proteins in the numerator and the proapoptotic protein Bax in the denominator, higher ratio values are observed with higher levels of the antiapoptotic proteins or lower levels of the proapoptotic proteins.

Pairwise associations between patient covariates were assessed graphically for pairs of numerical variables by examining scatterplots, by Wilcoxon-Mann-Whitney and Kruskal-Wallis (29) test statistics for categorical and continuous variables and by the Fisher exact test (30) and its generalizations (31) for pairs of categorical variables. Unadjusted survival and DFS analyses were performed using Kaplan-Meier plots (32) . Unadjusted comparisons of survival and DFS between patient subgroups were made using the log-rank test (33) . The Cox proportional hazards model (34) and its generalizations (35) were used to assess the ability of the treatment indicators including BAX, PKC{alpha}, BCL2, ratios of B2:BX or PK*B2:BX, and other patient characteristics (age, sex, bilirubin, performance status, presence of an AHD, fibrinogen, hemoglobin, platelet count, white blood count, and albumin) to predict survival and DFS. To assess possible threshold effects of BCL2 on survival and DFS while avoiding searching for optimal cutpoints of expression levels of BAX, PKC{alpha}, B2:BX, and PK*B2:BX, results were divided into quartiles (36) . When quartiles demonstrated similar outcomes, those arms were collapsed. Cox regression models for survival and DFS were obtained by initially identifying any important interactive effects between PKC{alpha}, BAX, BCL2, B2:BX, and PK*B2:BX, cytogenetics, treatment, and other patient characteristics, including these effects in an initial set of variables along with patient covariates, and then performing a backward elimination with a P cutoff of 0.05. Variables were selected for inclusion in the model based on their previous recognition as important prognostic factors (37, 38, 39) and evidence of association with survival in this population. To avoid colinearities, variables showing a strong association with PKC{alpha}, BAX, BCL2, B2:BX, PK*B2:BX, or cytogenetics were not included in the multivariate Cox model analyses. All computations were performed using Statistica version 5.1 M (StatSoft, Inc., Tulsa, OK).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Correlation of Expression of BAX and PKC{alpha} with Clinical Features of Patients.
Expression of BAX and PKC{alpha} were measured in 165 patient samples, and a detectable signal was observed in 161 and 163 samples, respectively. The median level and ranges of expression of BAX were similar in all FAB classifications analyzed with the exception of FAB M0 and FAB M4. These categories had narrower and broader ranges, respectively (Fig. 2)Citation . Similarly the median expression levels and range of BAX were similar in cells containing all cytogenetic abnormalities except inversion 16, where the samples displayed a lower median and a narrower range of expression. There were no differences in median PKC{alpha} expression or the range of expression among different FAB groups or cytogenetic categories. There was also no significant correlation between the level of expression of PKC{alpha}, BAX, or BCL2 and the percentage of blasts in the sample, gender, age, history of AHD, cytogenetics (divided into FIPC or UC), hemoglobin, platelets, albumin, serum bilirubin, or serum fibrinogen. Interestingly, there was a highly statistically significant correlation between PKC{alpha} and BAX expression (r = 0.30; P < 0.00001). Furthermore, levels of PKC{alpha} but not BAX correlated with WBC count (r = 0.29; P = 0.0001).



View larger version (25K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 2. Range of expression of BAX and PKC{alpha} in different FAB and cytogenetic categories. ?, patients with indeterminate FAB classification; IM, patients with insufficient metaphases. There were 10, 36, 34, 15, 41, 10, 5, 2, and 12 patients in FAB categories M0 through M?, respectively. There were 13 patients with t(8;21); 6 with inversion 16 (Inv16), 13 with t(15;17), 54 diploid (Diploid), 7 with IM, 24 miscellaneous (Misc), 30 with -5 or -7 (-5,-7), 11 with +8, 5 with 11q23, and 2 Ph1 patients. Bars, SD.

 
Neither BAX nor PKC{alpha} Expression Levels Alone Are Prognostic of Remission Induction Rate or Overall Survival.
To assess the possible prognostic impact of BAX or PKC{alpha} expression levels on the response to remission induction therapy or overall survival, the population was divided into quartiles. Because we had observed previously that cytogenetics affected the prognostic impact of BCL2, we evaluated the prognostic impact of BAX and PKC{alpha} among patients with FIPC and UC separately. Regardless of whether patients were divided into quartiles or halves or whether all patients or only those with FIPC or UC were included, the level of expression of BAX and PKC{alpha}, as determined, failed to correlate with the remission rate or overall survival (Table 2)Citation . However, when stratified by cytogenetics, patients with FIPC demonstrated increased survival with increasing BAX expression (Fig. 3)Citation , although this finding did not achieve statistical significance (P = 0.08).


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

  		Table 2 Effect of BAX, PKC{alpha}, B2:BX, PK*B2:BX, and cytogenetics on response to therapy and survival

 


View larger version (20K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 3. Effect of BAX expression on survival among patients with FIPC. The panel shows the Kaplan Meier survival curves of FIPC patients for each quartile of BAX expression. Lowest quartile (|, n = 50), second quartile ({diamondsuit}, n = 50), third quartile ({Delta}, n = 35), highest quartile ({diamond}, n = 30)

 
Ratios of B2:BX and PK*(B2:BX) Are Prognostic of Response to Therapy and Outcome But Are Dependent on Cytogenetics.
When all patients are grouped together, none of the three interactive terms B2:BX, PK*B2, or PK*B2:BX was predictive of achieving remission (Table 2)Citation or of overall survival. In stark contrast, both B2:BX and PK*B2:BX are strongly predictive of both response to induction therapy and survival when the patients are stratified by FIPC and UC. Initially patients were divided into quartiles based on the expression levels of the three variables, but response rates and survival curves for the two upper quartiles and the two lower quartiles were similar (data not shown), and hence they were collapsed into groups with ratios above and below the median for both B2:BX and PK*B2:BX. Patients with high B2:BX ratios behaved similarly regardless of cytogenetics, as shown (Fig. 4A),Citation but a low B2:BX ratio was a favorable finding for patients with FIPC but, paradoxically, a very adverse finding for patients with UC. Similarly, patients with a high PK*B2:BX ratio had similar response rates and outcomes regardless of cytogenetics, whereas those with low ratios did very well if they had FIPC but very poorly if they were in the UC group (Fig. 4B).Citation The PK*B2 term was not significantly prognostic and was not considered further. Similar results, albeit with higher Ps, are observed if the analysis was limited to patients with >25 or >50% blasts in the sample, suggesting that these results are not affected by the percentage of blasts in the sample.



View larger version (29K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 4. Effect of B2:BX and PK*(B2:BX) ratios on survival stratified by cytogenetics. The panels show Kaplan Meier survival curves for B2:BX (A) or PK*(B2:BX) (B) expression divided by whether expression was below (low ratio) or above (high ratio) the median expression for patients with either FIPC or UC. Samples size for A: FIPC and low ratio (n = 50), FIPC and high ratio (n = 50), UC and high ratio (n = 35), and UC and low ratio (n = 31). Samples size for B: FIPC and low ratio (n = 52), FIPC and high ratio (n = 49), UC and high ratio (n = 33), and UC and low ratio (n = 32). Fav & Int Cyto, FIPC; Unfav Cyto, UC.

 
B2:BX and PK*(B2:BX) Ratios Are Independent Prognostic Factors in Univariate and Multivariate Analysis.
Univariate analysis was performed using previously identified AML prognostic factors (listed in statistical methods) on BAX, PKC{alpha}, B2:BX, PK*B2:BX (above or below the median) and on four interactive variables that combined cytogenetics and the interactive terms: (a) FIPC and low B2:BX; (b) UC and low B2:BX; (c) FIPC and low PK*B2:BX; or (d) UC and low PK*B2:BX. Variables that were significant by univariate analysis included FAB M3, gender, age >50, history of AHD >2 months, performance status, favorable cytogenetics, UC, BCL2 quartile, and the four interactive variables described above.

Multivariate Cox models for survival that include treatment and the other patient characteristics, in addition to the B2:BX cytogenetics or the PK*B2:BX cytogenetics terms, are summarized in Table 3Citation . These models were obtained via the backward elimination procedure described in the methods. Results for DFS were similar and are not presented. Depending on the model used, all four of the B2:BX or PK*B2:BX cytogenetics variables were independent predictors of outcome, confirming the pattern indicated by the preliminary analyses. Either considered per se or covariate adjusted, patients with favorable cytogenetics and a more proapoptotic ratio of either B2:BX or PK*B2:BX had longer overall survival than those with more antiapoptotic, or protective, ratio regardless of cytogenetics. A proapoptotic ratio (high PK*B2:BX or high B2:BX) and unfavorable cytogenetics was a particularly unfavorable feature.


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

  		Table 3 Summary of multivariate analysis

Ps from multivariate analyses with survival as the endpoint including interactive variables for cytogenetics and either B2:BX or PK*(B2:BX) or PK*(BX:B2) ratios are shown.

 
The Rate of Spontaneous Apoptosis Does Not Differ among the Different Groups.
PARP is a substrate of caspases 3 and 9, and its cleavage is a marker for apoptosis. To test whether the apoptotic potential suggested by these ratios correlated with the rate of spontaneous apoptosis present in these cells, we assayed a subset of these patients (n = 50) for evidence of PARP cleavage by Western blot. Among patients with a proapoptotic ratios, cleaved PARP was detected in 1 of 14 FIPC and 2 of 14 UC cases. Among those with antiapoptotic ratios, 2 of 12 FIPC and 1 of 10 UC cases demonstrated PARP cleavage.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Previously, we and others have found that BCL2 expression correlated with remission induction rates and overall survival for patients with AML (14, 15, 16) .5 This study measured the expression levels of PKC{alpha} and Bax, two potential regulators of cell survival and apoptosis, in cells from untreated patients with AML. Expression of BAX and PKC{alpha} were found to be both ubiquitous and heterogeneous among all patients regardless of FAB classification or cytogenetic category, except for a narrow range of expression of BAX among patients with inversion 16. Furthermore, neither variable alone was prognostic of remission induction rates or overall survival. However, because both proteins may potentially modulate the function of the antiapoptotic gene product BCL2, we tested for any potential prognostic interactions by evaluating the interactive terms of each protein and BCL2. When expressed as an interactive variable, both PKC{alpha} and BAX proteins appear to modulate the prognostic impact of BCL2 in a cytogenetic-dependent fashion. However, we found no correlation between either BAX or PKC{alpha} and BCL2 expression among patient samples. The expectation would be that the prognostic impact of BCL2 alone would be forfeited when forming interactive terms from essentially random levels of either BAX or PKC{alpha} and BCL2. This would be especially true when forming an interactive variable from both PKC{alpha} and Bax and BCL2. Surprisingly, these interactive terms yielded greater prognostic discrimination than BCL2 by itself, suggesting that although the relationships were not immediately apparent based on raw expression levels, a functional relationship truly exists. The majority of the strength of this relationship derives from the B2:BX ratio (Fig. 4A),Citation but the addition of PKC{alpha} to the ratio increases the prognostic discrimination in both FIPC and UC patients (Fig. 4Citation , compare A and B).

Consequently, among FIPC or UC patients, either B2:BX or PK*B2:BX ratios are highly prognostic for both response to remission induction therapy as well as overall survival. Intuitively, a propensity for AML leukemic cells to undergo apoptosis, as defined by a "proapoptotic" ratio would seem to be a good prognostic feature. Indeed the "best" prognostic group consisted of FIPC patients with ratios suggestive of a proapo-ptotic potential, indicated by low B2:BX or low PK*B2:BX ratios (Tables 2Citation and 3)Citation . Alternatively, patients with an antiapo-ptotic ratio, suggested by high B2/BX or high PK*B2:BX ratios, formed an "intermediate" prognosis group characterized by lower remission rates and median survivals compared with the "best" prognosis group, regardless of their cytogenetics. However, a more "proapoptotic" ratio was not always shown to be a prognostically favorable finding because low ratios in combination with UC appeared to form the "worst" outcome group, characterized by significantly lower remission rates and shorter overall survival. The division into three groups and relationship toward apoptotic potential are summarized in Fig. 5Citation .



View larger version (25K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 5. Net apoptotic direction and cytogenetics defines three prognostic groups of patients. The presumed net apoptotic direction based on B2:BX or PK*B2:BX ratios and cytogenetics were used to divide patients into three prognostic groups based on response to therapy and survival outcome.

 
These assays measured relative levels rather than the true native apoptosis rate of these leukemic cells or the ability of chemotherapy to induce apoptosis. There may be incomplete association between these ratios and the apoptotic potential of the cells that may help to explain this apparent paradox in which increased proapoptotic potential is positively associated with FIPC while negatively associated with UC. We attempted to look for such a correlation by assaying for PARP cleavage as a measure of spontaneous apoptosis, but no difference was apparent. However, this may simply reflect the rapid clearance of apoptotic cells from the circulation by the reticuloendothelial system because apoptotic cells are not detected in high number in the circulation of AML patients undergoing induction chemotherapy either.6 To address this more formally, we have initiated a prospective study to measure the expression of these proteins by Western blot and FACS and are correlating this with the degree of apoptosis present spontaneously and after exposure to 1-ß-D-arabinofuranosylcytosine. Another caveat is that this study did not capture samples from patients with emergency presentations requiring urgent intervention. Not surprisingly, this cohort with more adverse prognostic features (typically including higher WBC counts, worse performance status, and frequently with serious infection at the time of diagnosis) did worse than the studied cohort. Whether this worse outcome derives from patient condition or a difference in the apoptotic susceptibility of their cells is undetermined.

This observation may be based, at least in part, on the prevailing qualitative or functional levels of BCL2 after posttranslational regulation such as its phosphorylation, which can be modulated by PKC{alpha}. However, whether BCL2 is phosphorylated at all or the extent of phosphorylation attributable to PKC{alpha} was not measured in these samples. There are numerous additional members of the BCL2 family of proteins as well as other proteins like those of the IAP family, which modify the message delivered by the BCL2 family that were not analyzed in this study. We have shown previously that levels of expression of caspases 2 and 3 are prognostic in AML (25) , and it is possible that there are additional prognostic effects imparted by downstream members of the apoptotic pathways. Studies to determine the amount of phosphorylated BCL2 in some of these samples and additional associations with downstream apoptosis regulators are future focuses of our laboratory. The localization of these proteins, cytosol versus mitochondria, may also affect the linkage between these ratios and actual apoptosis susceptibility.

If this relationship between apoptotic potential and cytogenetics is validated, it could have important implications for novel therapeutic strategies aimed at inducing apoptosis. Accurate determination of whether a block in apoptosis exists and where in the pathway it occurs would be required to effectively apply apoptosis-inducing therapy to patients in the intermediate and worst groups. For example, BCL2 antisense might be effective for intermediate group patients, where high levels of BCL2 suggest that the leukemic cells are dependent on BCL2 protection, but it would be expected to be ineffective for patients in the worst group, where levels of BCL2 are already relatively low. Patients with relatively high levels of mitochondrial PKC{alpha} and phosphorylated BCL2 might benefit from the selective inhibition of PKC{alpha} (perhaps by the drug UCN-01 (40 , 41) and blockade of BCL2 phosphorylation prior to institution of induction remission chemotherapy. The combination of conventional induction therapy with therapy directed at increasing the induction of apoptosis comprise a unique treatment strategy individually tailored to the apoptosis profile of a patient’s leukemic cells, which may improve response and outcome.


   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 NIH PO1Grant 55164-04 and Leukemia Society of America Grant 6089-99. Back

2 To whom requests for reprints should be addressed, at Section of Molecular Hematology and Therapy, M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Box 81, Houston, TX 77030-4095. Phone: (713) 794-1568; Fax: (713) 794-1938; E-mail: skornblau{at}mdacc.tmc.edu Back

3 The abbreviations used are: B2:BX, BCL2:BAX ratio; PK*B2, PKC{alpha}*BCL2; PKC{alpha}, protein kinase C {alpha}; AML, acute myelogenous leukemia; FIPC, favorable and intermediate prognosis cytogenetics; UC, unfavorable prognosis cytogenetics; PARP, poly(ADP-ribose) polymerase; DFS, disease-free survival; AHD, antecedent hematological disorder; FAB, French-American-British. Back

4 X. Fang and G. Mills, personal communication. Back

5 M. Andreeff, unpublished data. Back

Received 11/ 5/99; revised 12/27/99; accepted 12/28/99.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Yang E., Korsmeyer S. J. Molecular thanatopsis: a discourse on the BCL2 family and cell death. Blood, 88: 386-401, 1996.[Free Full Text]
  2. Cohen G. M. Caspases: the executioners of apoptosis. Biochem. J., 326: 1-16, 1997.
  3. Rowan S., Fisher D. E. Mechanisms of apoptotic cell death. Leukemia (Baltimore), 11: 457-465, 1997.[CrossRef][Medline]
  4. Kroemer G. The proto-oncogene bcl-2 and its role in regulating apoptosis. Nat. Med., 3: 614-620, 1997.[CrossRef][Medline]
  5. Webb S. J., Harrison D. J., Wyllie A. H. Apoptosis: an overview of the process and its relevance in disease. Adv. Pharmacol., 41: 1-34, 1997.
  6. Reed J. C. Bcl-2 family proteins: strategies for overcoming chemoresistance in cancer. Adv. Pharmacol., 41: 501-532, 1997.
  7. Konopleva, M., Zhao, S., Xie, Z., Seagall, H., Younes, A., Claxton, D. F., Estrov, Z., Kornblau, S. M., and Andreeff, M. Apoptosis. Molecules and mechanisms. In: A. J. P. Veerman, R. Pieters, and G. J. L. Kaspers (eds.), Drug Resistance in Leukemia and Lymphoma III. Amsterdam: Plenum Publishing Corp., 1999.
  8. Oltvai Z. N., Milliman C. L., Korsmeyer S. J. Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell, 74: 609-619, 1993.[CrossRef][Medline]
  9. May W. S., Tyler P. G., Ito T., Armstrong D. K., Qatasha K. A., Davidson N. E. Interleukin-3 and bryostatin-1 mediate hyperphosphorylation of BCL2 {alpha} in association with suppression of apoptosis. J. Biol. Chem., 269: 26865-26870, 1994.[Abstract/Free Full Text]
  10. Ito T., Deng X., Carr B., Strauss E. Bcl-2 phosphorylation required for anti-apoptosis function. J. Biol. Chem., 272: 11671-11673, 1997.[Abstract/Free Full Text]
  11. Ruvolo P. P., Deng X., Carr B. K., May W. S. A functional role for mitochondrial protein kinase C-{alpha} in bcl2 phosphorylation and suppression of apoptosis. J. Biol. Chem., 273: 25436-25442, 1998.[Abstract/Free Full Text]
  12. del Peso L., Gonzalez-Garcia M., Page C., Herrera R., Nunez G. Interleukin-3-induced phosphorylation of BAD through the protein kinase Akt. Science (Washington DC), 278: 687-689, 1997.[Abstract/Free Full Text]
  13. Maung Z. T., MacLean F. R., Reid M. M., Pearson A. D. J., Proctor S. J., Hamilton P. J., Hall A. G. The relationship between bcl-2 expression and response to chemotherapy in acute leukaemia. Br. J. Haematol., 88: 105-109, 1994.[Medline]
  14. Campos L., Rouault J-P., Sabido O., Oriol P., Roubi N., Vasselon C., Archimbaud E., Magaud J-P., Guyotat D. High expression of bcl-2 protein in acute myeloid leukemia cells is associated with poor response in chemotherapy. Blood, 81: 3091-3096, 1993.[Abstract/Free Full Text]
  15. Lauria F., Raspador D., Rondelli D., Ventura M. A., Facchini M., Visani G., Forconi F., Tura S. High bcl-2 expression in acute myeloid leukemia cells correlates with CD34 positivity and complete remission rate. Leukemia (Baltimore), 11: 2075-2078, 1997.[CrossRef][Medline]
  16. Karakas T., Maurer U., Weidmann E., Miething C. C., Hoelzer D., Bergmann L. High expression of bcl-2 mRNA as a determinant of poor prognosis in acute myeloid leukemia. Ann. Oncol., 9: 159-165, 1998.[Abstract/Free Full Text]
  17. Kornblau S. M., Thall P. F., Estrov Z., Walterscheid M., Patel S., Theriault A., Keating M. J., Kantarjian H., Estey E., Andreeff M. The prognostic impact of BCL2 protein expression in acute myelogenous leukemia varies with cytogenetics. Clin. Cancer Res., 5: 1758-1766, 1999.[Abstract/Free Full Text]
  18. Lepelley P., Soenen V., Preudhomme C., Merlat A., Cosson A., Fenaux P. bcl-2 expression in myelodysplastic syndromes and its correlation with hematological features, p53 mutations and prognosis. Leukemia (Baltimore), 9: 726-730, 1995.[Medline]
  19. Charpin C., Garcia S., Bonnier P., Martini F., Andrac L., Horschowski N., Lavaut M-N., Allasia C. bcl-2 automated and quantitative immunocytochemical assays in breast carcinomas: correlation with 10-year follow-u. p. J. Clin. Oncol., 16: 2025-2031, 1998.[Abstract]
  20. Campana D., Coustan-Smith E., Manabe A., Buschle M., Raimondi S. C., Behm F. G., Ashmun R., Arico M., Biondi A., Pui C-H. Prolonged survival of B-lineage acute lymphoblastic leukemia cells is accompanied by overexpression of bcl-2 protein. Blood, 81: 1025-1031, 1993.[Abstract/Free Full Text]
  21. Coustan-Smith E., Kitanaka A., Pui C-H., McNinch L., Evans W. E., Raimondi S. C., Behm F. G., Arico M., Campana D. Clinical relevance of bcl-2 overexpression in childhood acute lymphoblastic leukemia. Blood, 87: 1140-1146, 1996.[Abstract/Free Full Text]
  22. Volm M., Zintl F., Edler L., Sauerbrey A. Prognostic value of protein kinase C, proto-oncogene products and resistance-related proteins in newly diagnosed childhood acute lymphoblastic leukemia. Med. Pediatr. Oncol., 28: 117-126, 1998.
  23. Kornblau S. M., Andreeff M., Hu S-X., Xu H-J., Patel S., Theriault A., Koller C., Kantarjian H., Estey E. H., Deisseroth A. B., Benedict W. F. Low and maximally phosphorylated levels of the retinoblastoma protein confer poor prognosis in newly diagnosed acute myelogenous leukemia: a prospective study. Clin. Cancer Res., 4: 1955-1963, 1998.[Abstract]
  24. Estrov Z., Thall P., Talpaz M., Estey E. H., Kantarjian H., Harris D., Van Q., Walterscheid M., Kornblau S. M. Caspase 2 and caspase 3 protein levels as predictors for survival in acute myelogenous leukemia. Blood, 92: 3090-3097, 1998.[Abstract/Free Full Text]
  25. Le X. F., Groner Y., Levanon D., Kornblau S., Hittelman W., Arlinghaus R. B., Chang K. S. AML2, a nuclear phosphoprotein with a possible role in myeloid differentiation and leukemogenesis. Blood, 90(Suppl.1): 46a 1997.
  26. Le X. F., Claxton D., Kornblau S., Fan Y., Mu Z. M., Chang K. S. Characterization of the ETO and AML1-ETO proteins involved in 8;21 translocation in acute myelogenous leukemia. Eur. J. Haematol., 60: 217-225, 1998.[Medline]
  27. Claxton, D., Xie, Q., Patel, S., Deisseroth, A. B., and Kornblau, S. M. The gene product of the CBFB-MYH11. Leukemia (Baltimore), 1479–1485, 1996.
  28. Deng X., Ito T., Carr B., Mumby M., May W. S. Reversible phosphorylation of BCL2 following IL-3 or bryostatin 1 is mediated by the direct interaction with protein phosphatase 2a. J. Biol. Chem., 273: 34157-34163, 1998.[Abstract/Free Full Text]
  29. Snedecor, G. W., and Cochran, W. G. Statistical Methods, Ed. 7. Iowa: Iowa State University Press, 1980.
  30. Fisher R. A. The condition under which {chi}2 measures the discrepancy between observation and hypothesis. J. R. Stat. Soc., 87: 442-450, 1924.
  31. Mehta C. R. The exact analysis of contingency tables in medical research. Stat. Methods Med. Res., 3: 135-156, 1994.[Medline]
  32. Kaplan E. L., Meier P. Nonparametric estimation from incomplete observation. J. Am. Stat. Assoc., 53: 457-481, 1958.[CrossRef]
  33. Mantel N. Evaluation of survival data and two new rank order statistics arising in its consideration. J. R. Stat. Soc., 50: 63-170, 1966.
  34. Cox D. R. Regression models and life tables. J. R. Stat. Soc., 34: 187-220, 1972.
  35. Fleming, T. R., and Harrington, D. P. Counting Processes and Survival Analysis. New York: John Wiley & Sons, Inc., 1991.
  36. Altman D. G., Lausen B., Sauerbrei W., Schumacher M. Dangers of using "optimal" cutpoints in the evaluation of prognostic factors. J. Natl. Cancer Inst., 86: 829-835, 1994.[Free Full Text]
  37. Schiffer C. A., Lee E., Tomiyasu T., Wiernik P., Testa J. Prognostic impact of cytogenetic abnormalities in patients with de novo acute nonlymphocytic leukemia. Blood, 73: 263-270, 1989.[Abstract/Free Full Text]
  38. Smith T. L., McCredie K. Prediction of survival during induction therapy in patients with newly diagnosed acute myeloblastic leukemia. Leukemia (Baltimore), 3: 257-263, 1989.[Medline]
  39. Keating M. J., Cork A., Broach Y., Smith T., Walters R. S., McCredie K. B., Trujillo J., Freireich E. J. Toward a clinically relevant cytogenetic classification of AML. Leuk. Res., 11: 119-133, 1987.[CrossRef][Medline]
  40. Kawakami K., Futami H., Takahara J., Yamaguchi K. UCN-01, 7-hydroxyl-staurosporine, inhibits kinase activity of cyclin-dependent kinases and reduces the phosphorylation of the retinoblastoma susceptibility gene product in A549 human lung cancer cell line. Biochem. Biophys. Res. Commun., 219: 778-783, 1996.[CrossRef][Medline]
  41. Shao R. G., Shimizu T., Pommier Y. 7-Hydroxystaurosporine (UCN-01) induces apoptosis in human colon carcinoma and leukemia cells independent of p53. Exp. Cell Res., 234: 388-397, 1997.[CrossRef][Medline]



This article has been cited by other articles:


Home page
 BloodHome page
J. Greiner, M. Schmitt, L. Li, K. Giannopoulos, K. Bosch, A. Schmitt, K. Dohner, R. F. Schlenk, J. R. Pollack, H. Dohner, et al.
Expression of tumor-associated antigens in acute myeloid leukemia: implications for specific immunotherapeutic approaches
Blood, December 15, 2006; 108(13): 4109 - 4117.
[Abstract] [Full Text] [PDF]

Home page
 Molecular Cancer TherapeuticsHome page
R. Tibes, Y. Qiu, Y. Lu, B. Hennessy, M. Andreeff, G. B. Mills, and S. M. Kornblau
Reverse phase protein array: validation of a novel proteomic technology and utility for analysis of primary leukemia specimens and hematopoietic stem cells.
Mol. Cancer Ther., October 1, 2006; 5(10): 2512 - 2521.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
J. E. Karp
Leukemia does not live by 1 lesion alone
Blood, October 1, 2006; 108(7): 2133 - 2134.
[Full Text] [PDF]

Home page
 BloodHome page
S. M. Kornblau, M. Womble, Y. H. Qiu, C. E. Jackson, W. Chen, M. Konopleva, E. H. Estey, and M. Andreeff
Simultaneous activation of multiple signal transduction pathways confers poor prognosis in acute myelogenous leukemia
Blood, October 1, 2006; 108(7): 2358 - 2365.
[Abstract] [Full Text] [PDF]

Home page
 J. Cell Sci.Home page
L. Saraiva, R. D. Silva, G. Pereira, J. Goncalves, and M. Corte-Real
Specific modulation of apoptosis and Bcl-xL phosphorylation in yeast by distinct mammalian protein kinase C isoforms
J. Cell Sci., August 1, 2006; 119(15): 3171 - 3181.
[Abstract] [Full Text] [PDF]

Home page
 CarcinogenesisHome page
A. F. Badawi, Y. Liu, M. B. Eldeen, W. Morrow, Z. R. Razak, M. Maradeo, and M. Z. Badr
Age-associated changes in the expression pattern of cyclooxygenase-2 and related apoptotic markers in the cancer susceptible region of rat prostate
Carcinogenesis, September 1, 2004; 25(9): 1681 - 1688.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
A. F. Badawi, M. B. Eldeen, Y. Liu, E. A. Ross, and M. Z. Badr
Inhibition of Rat Mammary Gland Carcinogenesis by Simultaneous Targeting of Cyclooxygenase-2 and Peroxisome Proliferator-activated Receptor {gamma}
Cancer Res., February 1, 2004; 64(3): 1181 - 1189.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
B. Z. Carter, S. M. Kornblau, T. Tsao, R.-Y. Wang, W. D. Schober, M. Milella, H.-G. Sung, J. C. Reed, and M. Andreeff
Caspase-independent cell death in AML: caspase inhibition in vitro with pan-caspase inhibitors or in vivo by XIAP or Survivin does not affect cell survival or prognosis
Blood, December 1, 2003; 102(12): 4179 - 4186.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
A. D. Schimmer, I. M. Pedersen, S. Kitada, E. Eksioglu-Demiralp, M. D. Minden, R. Pinto, K. Mah, M. Andreeff, Y. Kim, W. S. Suh, et al.
Functional Blocks in Caspase Activation Pathways Are Common in Leukemia and Predict Patient Response to Induction Chemotherapy
Cancer Res., March 15, 2003; 63(6): 1242 - 1248.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
G. Del Poeta, A. Venditti, M. I. Del Principe, L. Maurillo, F. Buccisano, A. Tamburini, M. C. Cox, A. Franchi, A. Bruno, C. Mazzone, et al.
Amount of spontaneous apoptosis detected by Bax/Bcl-2 ratio predicts outcome in acute myeloid leukemia (AML)
Blood, March 15, 2003; 101(6): 2125 - 2131.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
M. Milella, Z. Estrov, S. M. Kornblau, B. Z. Carter, M. Konopleva, A. Tari, W. D. Schober, D. Harris, C. E. Leysath, G. Lopez-Berestein, et al.
Synergistic induction of apoptosis by simultaneous disruption of the Bcl-2 and MEK/MAPK pathways in acute myelogenous leukemia
Blood, May 1, 2002; 99(9): 3461 - 3464.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
J. A. Vrana, C. K. Bieszczad, E. S. Cleaveland, Y. Ma, J. P. Park, T. K. Mohandas, and R. W. Craig
An MCL1-overexpressing Burkitt Lymphoma Subline Exhibits Enhanced Survival on Exposure to Serum Deprivation, Topoisomerase Inhibitors, or Staurosporine but Remains Sensitive to 1-{beta}-D-Arabinofuranosylcytosine
Cancer Res., February 1, 2002; 62(3): 892 - 900.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
M. Konopleva, T. Tsao, P. Ruvolo, I. Stiouf, Z. Estrov, C. E. Leysath, S. Zhao, D. Harris, S. Chang, C. E. Jackson, et al.
Novel triterpenoid CDDO-Me is a potent inducer of apoptosis and differentiation in acute myelogenous leukemia
Blood, January 1, 2002; 99(1): 326 - 335.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
A. D. Schimmer, D. W. Hedley, L. Z. Penn, and M. D. Minden
Receptor- and mitochondrial-mediated apoptosis in acute leukemia: a translational view
Blood, December 15, 2001; 98(13): 3541 - 3553.
[Full Text] [PDF]

Home page
 J Natl Cancer Inst MonogrHome page
X. Deng, S. M. Kornblau, P. P. Ruvolo, and W. S. May Jr.
Regulation of Bcl2 Phosphorylation and Potential Significance for Leukemic Cell Chemoresistance
J Natl Cancer Inst Monographs, December 1, 2000; 2000(28): 30 - 37.
[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