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(American Journal of Pathology. 1999;155:955-965.)
© 1999 American Society for Investigative Pathology

Regular Articles

Overexpression of bax Associated with Mutations in the Loop-Sheet-Helix Motif of p53

Cheng-long Huang*, Nobuoki Kohno{dagger}, Haruhiko Inufusa{ddagger}, Ken Kodama§, Toshihiko Taki* and Masayuki Miyake*

From the Department of Thoracic Surgery and Department V of Oncology,*
Kitano Hospital, Tazuke Kofukai Medical Research Institute, Osaka, Japan; the Second Department of Internal Medicine,{dagger}
Ehime University School of Medicine, Ehime, Japan; the First Department of Surgery,{ddagger}
Kinki University School of Medicine, Osaka, Japan; and the Department of Surgery,§
Osaka Medical Center for Cancer and Cardiovascular Diseases, Osaka, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Recent investigations have revealed that mutations of the loop-sheet-helix motif of p53 is a significant factor for a poor prognosis in patients with non-small-cell lung cancer (NSCLC). To clarify this mechanism, bcl-2 and bax expression were evaluated in relation to mutations of p53. Tumor tissues of 203 patients with NSCLC were analyzed. Immunohistochemistry was performed to evaluate bcl-2 and bax expression, and polymerase chain reaction single-strand conformation polymorphism following direct sequencing was performed to investigate p53 status. A total of 79 carcinomas were bcl-2 positive, 146 carcinomas were bax positive, and 72 carcinomas had missense mutations of p53. There was no difference in bcl-2 expression in relation to p53 status. On the other hand, tumors with structural mutations of p53 had significantly lower expression of bax than those with wild-type p53 (P = 0.0026). In contrast, tumors with mutations of the loop-sheet-helix motif of p53 had significantly higher expression of bax than those with wild-type p53 (P = 0.0236). The frequency of a bcl-2/bax ratio of >=1 was significantly lower in tumors with mutations of the loop-sheet-helix motif than that in tumors with wild-type p53 (P = 0.0240). The bcl-2/bax ratio status was a significant factor for a prognosis in patients with NSCLC (P = 0.0083). Mutations of the loop-sheet-helix motif of p53 were correlated with overexpression of bax, while other mutations of p53 were correlated with low levels of bax expression. This variation in pattern of bax expression in relation to mutant p53 might reflect the biological behavior of tumors in patients with bcl-2-positive NSCLC.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
p53 is one of the major tumor suppressor genes,1-4 and most nonfunctioning p53 proteins with missense mutations of one p53 allele coupled to deletion of the second allele seem to cause carcinogenesis.5 However, many studies have reported that some mutant p53 genes could cause not only loss of normal function but also new carcinogenesis.6-8 p53 protein is a DNA-binding protein, with a sequence-specific DNA-binding region in the core domain.9,10 Two regions of the core domain of the p53 protein, the loop-sheet-helix motif (codons 273 to 286) and the L3-loop (codons 237 to 250), are considered to play important roles in contact with target DNA.11 Recently, we have found that the mutation of the loop-sheet-helix motif of p53 is a significant factor for a poor prognosis in patients with non-small-cell lung cancer (NSCLC).12 Similarly, mutations in these two regions of p53 were also reported to have a strong effect on clinical outcome in head and neck cancer.13 These results suggested that mutations of these regions of p53 could not control their target genes and precisely reflect the biological behavior of these types of malignant tumors.

To date, genes considered to be target genes of p53 include bcl-2,14 bax,15 topoisomerase II,16 insulin-like growth factor binding protein-3 (IGF BP-3),17 vascular endothelial growth factor (VEGF),18 and multidrug resistance (MDR).19 Since many NSCLCs are resistant to radio-chemotherapy in themselves, the poor prognosis of patients with mutations of the loop-sheet-helix motif of p53 might be associated with cell cycle regulation and apoptosis more than with radio-chemoresistance.5 In addition, recent studies have demonstrated that bcl-2 and bax, among members of the bcl-2 family, regulate not only apoptosis but also the cell cycle.20-24 Therefore, we performed the additional study focusing on bcl-2 and bax expression in relation to mutations of p53 in patients with NSCLC to clarify the mechanism of clinical behavior of these mutations. We also classified the mutations of p53 into three groups as described previously; mutations from codon 273 to codon 286 were classified as mutations of the loop-sheet-helix motif, mutations from codon 237 to codon 250 as mutations of the L3-loop, and mutations of other regions as structural mutations.13,25 In addition, because bax is widely expressed in normal tissues, such as smooth muscle cells and the bronchial wall,26 results by Northern blotting or quantitative reverse transcriptase polymerase chain reaction (RT-PCR) could be confused by bax expression in surrounding normal tissues, and blind RNA analysis was not suitable for this study. Therefore, immunohistochemistry was performed to evaluate bcl-2 and bax expression in this study.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Clinical Characteristics of Patients

From January 1991 to April 1995, the patients with NSCLC who underwent surgery at the Department of Surgery of the Osaka Medical Center for Cancer and Cardiovascular Diseases and the Department of Thoracic Surgery of the Kitano Hospital, Medical Research Institute of Osaka, Japan, were studied as described in our previous report.12 Among these 204 patients, 203 cases were investigated, except one case with wild-type p53 whose specimen was not available for immunohistochemistry. They included 121 patients with adenocarcinoma, 71 patients with squamous cell carcinoma, and 11 patients with large-cell carcinoma. Tumor-node-metastasis (TNM) staging designations were made according to the postsurgical pathological international staging system.27 Patients' clinical records and histopathological diagnoses were fully documented. This report includes follow-up data as of February 28 in 1998. The median follow-up period for all patients was 41.8 ± 23.6 months.

All patients with N2 or N3 status had mediastinal radiotherapy (50 Gy in 25 fractions for a period of 5 weeks) after surgical resection and then were treated by two cycles of adjuvant chemotherapy including cisplatin (80 mg/m2 given intravenously on day 1) and vindesine (4 mg/m2 given intravenously on days 1, 8, and 15). Eighteen patients with T3 or T4 status, who were found to have incomplete resection, also received radiotherapy (50 Gy in 25 fractions for a period of 5 weeks) for microscopic-positive margins. The other patients had no radiotherapy before recurrence. Post-operative adjuvant systemic chemotherapy was given according to nodal status. Of 82 node-positive patients, 78 patients underwent treatment by the adjuvant chemotherapy with two cycles of cisplatin and vindesine. Of 121 node-negative patients, 118 patients did not have further adjuvant treatment. We detected distant metastases in 83 patients during the observation period, and among these 83 patients, 14 patients had also local recurrences. Seven patients had locoregional recurrences. After recurrence, locoregional tumors or lymph nodes were principally treated with radiotherapy. Patients with distant metastases were treated with other regimes of chemotherapy, including cisplatin and etoposide.

Immunohistochemistry

For immunohistochemistry of bcl-2 and bax, a mouse monoclonal antibody for bcl-2 (clone 124, Dako, Glostrup, Denmark) diluted at 1:50 and a rabbit polyclonal antibody for bax (N-20, Santa Cruz Biotechnology, Santa Cruz, CA) diluted at 1:100 were used. Formalin-fixed paraffin-embedded tissue was cut in 4-µm sections and mounted on poly-L-lysine-coated slides. Sections were deparaffinized and rehydrated. The slides were then heated in a microwave for 10 minutes in a 10-µmol/L citrate buffer solution at pH 6.0 and cooled to room temperature for 20 minutes. After quenching the endogenous peroxidase activity with 0.3% H2O2 (in absolute methanol) for 30 minutes, the sections were blocked for 2 hours at room temperature with 5% bovine serum albumin. Subsequently, duplicate sections were incubated overnight with the primary specific antibodies detecting bcl-2 and bax, respectively. Slides were then incubated for 1 hour with biotinylated anti-mouse IgG (Vector Laboratories, Burlingame, CA) for bcl-2 or biotinylated anti-rabbit IgG (Vector Laboratories) for bax. The sections were incubated with the avidin-biotin-peroxidase complex (Vector) for 1 hour, and the antibody binding was visualized with 3,3'-diaminobenzidine tetrahydrochloride. Finally, the sections were lightly counterstained with Mayer's hematoxylin (Figure 1) .



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  		Figure 1. Immunohistochemical staining of human non-small-cell lung cancer tissues using the avidin-biotin-peroxidase complex procedure. Original magnification, x200. A: Adenocarcinoma with positive bcl-2 expression. B: Squamous cell carcinoma with positive bcl-2 expression. C: Adenocarcinoma with positive bax expression. D: Squamous cell carcinoma with positive bax expression.

 
Positive staining of lymphocytes infiltrating into the tumor provided an internal control for bcl-2 staining. For bax staining, the internal control was provided by smooth muscle cells in the media of arterioles and in the bronchial wall.26 SP2 supernatants were used as negative controls for both antibodies.

All of the immunostained sections were reviewed by two pathologists who had no knowledge of the patients' clinical status. In cases of multiple areas of low intensity, five areas selected at random were scored, and in sections where all of the staining appeared intense, one random field was selected. The proportion of high- and low-staining tumor cells in each selected field was determined by counting individual tumor cells at high magnification. At least 200 tumor cells were scored per 40x field. All sections were scored in a semiquantitative fashion according to the method described previously, which considers both the intensity and percentage of cells staining at each intensity.28,29 Intensities were classified as 0 (no staining), +1 (weak staining), +2 (distinct staining), or +3 (very strong staining) in relation to the internal control described above.26 For each slide, a value designated as the HSCORE was obtained by application of the following algorithm: HSCORE = {Sigma} (I x PC), where I and PC represent staining intensity and percentage of cells that stain at each intensity, respectively, and corresponding HSCOREs were calculated separately. When the HSCORE in a given specimen was >=50, the sample was classified as bcl-2 positive or bax positive, and when the HSCORE was <50, the samples was classified as negative. In addition, the bcl-2 HSCORE was divided by the bax HSCORE in each tumor to obtain a bcl-2/bax ratio as described previously.29

PCR-SSCP and Sequencing

To investigate the mutations of p53, we performed PCR single-strand conformation polymorphism (PCR-SSCP) and direct sequencing as described in our previous report.12 Briefly, genomic DNA was extracted from frozen specimens by using proteinase K digestion and phenol/chloroform extraction. After 40 cycles of PCR amplification of exons 5 to 8 of p53 were performed, electrophoresis for SSCP was done to detect mutant bands that were stained with ethidium bromide and visualized under ultraviolet light. Then, to ascertain base changes and exclude the non-missense mutations detected by PCR-SSCP, direct sequencing was performed.

Statistical Analysis

Overall cancer-specific survival was defined from the date of operation to the date of cancer-related death. The statistical differences in bcl-2 and bax expression and mutations of p53 in relation to several clinical and pathological parameters were assessed by the {chi}2 test and the t-test. Moreover, the statistical significance of bcl-2 and bax expression among each mutant region of p53 was also analyzed by the t-test and the {chi}2 test. The Kaplan-Meier method was used to estimate the probability of overall survival as a function of time, and differences in the survival of subgroups of patients were compared with Mantel's log-rank test.30,31 Multivariate analyses were performed using the Cox regression model to study the effects of different variables on survival.32 All P values were based on two-tailed statistical analysis, and a P value <0.05 was considered to indicate statistical significance.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of bcl-2

Of the 203 tumors we studied, 79 carcinomas (38.9%) were bcl-2 positive, and bcl-2 expression was always diffuse in the cytoplasm (Table 1 and Figure 1, A and B ). The HSCORE of bcl-2 was 30.6 ± 61.8 in adenocarcinomas, 65.1 ± 81.4 in squamous cell carcinomas, and 95.9 ± 134.2 in large-cell carcinomas. The HSCORE of bcl-2 in adenocarcinomas was significantly lower than that in squamous cell carcinomas and that in large-cell carcinomas (P = 0.0011 and P = 0.0037, respectively). Of the 121 adenocarcinomas, 35 tumors (28.9%) were bcl-2 positive. Of the 71 squamous cell carcinomas, 39 tumors (54.9%) were bcl-2 positive. Of the 11 large-cell carcinomas, 5 tumors (45.5%) were bcl-2 positive (Table 1) . The frequency of bcl-2-positive tumors in squamous cell carcinomas was significantly higher than that in adenocarcinomas (P = 0.0016). However, there was no significant correlation between bcl-2 staining and other patient prognostic factors such as tumor status, nodal status, smoking habits, or tumor differentiation.


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  		Table 1. Distribution of bcl-2 and bax Status in 203 Patients with NSCLC According to Clinicopathological Characteristics

 
Expression of bax

One hundred and forty-six carcinomas (71.9%) had positive expression of bax, and bax expression was also always diffuse in the cytoplasm (Table 1 and Figure 1, C and D ). The HSCORE of bax was 108.6 ± 77.7 in adenocarcinomas, 83.9 ± 53.7 in squamous cell carcinomas, and 57.3 ± 66.3 in large-cell carcinomas. The HSCORE of bax in adenocarcinomas was significantly higher than that in squamous cell carcinomas and that in large-cell carcinomas (P = 0.0189 and P = 0.0360, respectively). Ninety-one tumors (75.2%) were bax positive in adenocarcinomas, fifty-one tumors (71.8%) were bax positive in squamous cell carcinomas, and four tumors (36.4%) were bax positive in large-cell carcinomas (Table 1) . The frequency of bcl-2-positive tumors in large-cell carcinomas was significantly lowest (P = 0.0231), whereas no difference in bax expression was found between adenocarcinomas and squamous cell carcinomas. There was no significant correlation between bax expression and other patient prognostic factors such as tumor status, nodal status, smoking habits, or tumor differentiation.

Mutations of p53

Of the 203 patients with NSCLC studied, 75 carcinomas (36.9%) had mutations of p53, including 72 carcinomas with missense mutations and 3 carcinomas with non-missense mutations (Table 2) . With respect to the mutant region of the p53 gene of these 72 carcinomas with missense mutations, 42 carcinomas had structural mutations, 15 carcinomas had mutations of the L3-loop, and 15 carcinomas had mutations of the loop-sheet-helix motif.


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  		Table 2. Characteristics of the Patients with Mutations of p53 in NSCLC

 
Expression of bcl-2 and bax in Relation to Mutant Region of p53

The HSCORE of bcl-2 was 42.8 ± 71.8 in tumors with wild-type p53, 53.9 ± 87.1 in tumors with structural mutations, 57.3 ± 91.1 in tumors with mutations of the L3-loop, and 42.7 ± 76.7 in tumors with mutations of the loop-sheet-helix motif. There was no difference in bcl-2 expression in relation to the mutant region of p53.

The HSCORE of bax was 106.5 ± 70.0 in tumors with wild-type p53, 70.1 ± 57.1 in tumors with structural mutations, 38.3 ± 39.9 in tumors with mutations of the L3-loop, and 150.7 ± 78.6 in tumors with mutations of the loop-sheet-helix motif (Figure 2) . Tumors with structural mutations had lower levels of bax expression than those with wild-type p53 (P = 0.0026), and tumors with mutations of the L3-loop also had lower levels of bax expression than those with wild-type p53 (P = 0.0003). In contrast, tumors with mutations of the loop-sheet-helix motif had higher levels of bax expression than those with wild-type p53 (P = 0.0236).



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  		Figure 2. HSCORE of bax in 203 patients with non-small-cell lung cancer in relation to p53 status. Bars indicate the standard error of each group.

 
With respect to the bcl-2/bax ratio in tumors, the frequency of a bcl-2/bax ratio of >=1 in relation to the mutant region of p53 was analyzed. Of 131 tumors (26.0%) with wild-type p53, 34 had a bcl-2/bax ratio >= 1, 15 of 42 tumors (35.7%) with structural mutations had a bcl-2/bax ratio >= 1, 6 of 15 tumors (40.0%) with mutations of the L3-loop had a bcl-2/bax ratio >= 1, and only 1 of 15 tumors (6.7%) with mutations of the loop-sheet-helix motif had a bcl-2/bax ratio >= 1 (Figure 3A) . The frequency of a bcl-2/bax ratio >= 1 in tumors with mutations of the loop-sheet-helix was significantly lower than that in any other subgroups (P = 0.0240).



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  		Figure 3. A: bcl-2/bax ratio of 203 patients with non-small-cell lung cancer in relation to p53 status. B: bcl-2/bax ratio of 79 patients with bcl-2-positive non-small-cell lung cancer in relation to p53 status.

 
In addition, 79 tumors with bcl-2-positive expression were classified into two groups in relation to the bcl-2/bax ratio. Of these 79 bcl-2-positive tumors, 34 of 49 tumors (69.4%) with wild-type p53 had a bcl-2/bax ratio >= 1, 15 of 17 tumors (88.2%) with structural mutations had a bcl-2/bax ratio >= 1, all of 6 tumors with mutations of the L3-loop had a bcl-2/bax ratio >= 1, and only 1 of 7 tumors (14.3%) with mutations of the loop-sheet-helix motif had a bcl-2/bax ratio >= 1 (Figure 3B) . Of the bcl-2-positive tumors, the frequency of a bcl-2/bax ratio >= 1 in tumors with mutations of the loop-sheet-helix was significantly lower than that in tumors with wild-type p53 (P = 0.0087).

Association of bcl-2 and bax Expression with Survival of NSCLC Patients

The survival of 203 patients with NSCLC stratified according to bcl-2, bax, and bcl-2/bax ratio status is shown in Table 3 and Figure 4 . The 5-year survival rate of patients with bcl-2-positive tumors was significantly better than that with bcl-2-negative tumors (74.5% versus 46.6%, P = 0.0006; Table 3 and Figure 4A ). Especially, of the 121 adenocarcinomas, patients with bcl-2-positive tumors had a better prognosis than those with bcl-2-negative tumors (74.1% versus 43.4%, 5-year survival, P = 0.0039). On the other hand, there was no difference in survival in relation to bax expression (56.5% versus 59.8%, 5-year survival; Table 3 and Figure 4B ).


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  		Table 3. Five-Year Survival of 203 Patients with NSCLC According to Clinicopathological Characteristics and Gene Status

 


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  		Figure 4. A: Overall survival of 203 patients with non-small-cell lung cancer in relation to bcl-2 status. B: Overall survival of 203 patients with non-small-cell lung cancer in relation to bax status. C: Overall survival of 203 patients with non-small-cell lung cancer in relation to bcl-2 and bax status. D: Overall survival of 203 patients with non-small-cell lung cancer in relation to bcl-2/bax ratio status.

 
The survival of 203 patients with NSCLC was then analyzed in relation to bcl-2 and bax status (Figure 4C) ; 56 patients had tumors positive for both bcl-2 and bax, 23 patients had bcl-2-positive but bax-negative tumors; 90 patients had bcl-2-negative and bax-positive tumors; and 34 patients had tumors negative for both bcl-2 and bax. Overall survival of patients with bcl-2-positive and bax-negative tumors was significantly better than that with tumors positive for both (91.3% versus 67.4%, 5-year survival, P = 0.0417). In addition, overall survival of patients with tumors positive for both was significantly better than that of patients with bcl-2-negative and bax-positive tumors (67.4% versus 49.9%, 5-year survival, P = 0.0341) and was also significantly better than that with tumors negative for both (67.4% versus 39.7%, 5-year survival, P = 0.0201). Furthermore, of the bcl-2-negative tumors, there was no difference in survival of patients in relation to bax expression.

With respect to the bcl-2/bax ratio, overall survival of patients with tumors having a bcl-2/bax ratio of >=1 was significantly better than that of patients with tumors with a bcl-2/bax ratio of <1 (81.0% versus 48.7%, 5-year survival, P = 0.0004; Table 3 and Figure 4D ). Of the 121 adenocarcinomas, overall survival of patients with a bcl-2/bax ratio >=1 was significantly better than that of patients with tumors with a bcl-2/bax ratio <1 (77.5% versus 46.0%, 5-year survival, P = 0.0131). In addition, this was similarly true of 71 patients with squamous cell carcinomas (84.4% versus 54.3%, 5-year survival, P = 0.0162).

Cox regression analysis of prognostic variables for NSCLC, adenocarcinoma, and squamous cell carcinoma, using both bcl-2 status and bcl-2/bax ratio status, is shown in Table 4 . The bcl-2/bax ratio status was a significant indicator for survival of patients with NSCLC (Hazard ratio = 3.290, P = 0.0083), those with adenocarcinoma (Hazard ratio = 3.242, P = 0.0137), and those with squamous cell carcinoma (Hazard ratio = 6.610, P = 0.0053), whereas bcl-2 status alone was not a significant factor for survival of these patients.


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  		Table 4. Multivariate Regression Analysis in Predicting Survival of 203 Patients with NSCLC

 
Association of bcl-2 Expression with Survival of NSCLC Patients According to Mutant Region of p53

Of patients with bcl-2-positive tumors, the 5-year survival of patients with mutations of the loop-sheet-helix motif of p53 was significantly lower than that of patients with wild-type p53 (28.6% versus 75.0%, P = 0.0050) and that of patients with structural mutation of p53 (28.6% versus 87.8%, P = 0.0082; Figure 5 ).



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  		Figure 5. Overall survival of 79 patients with bcl-2-positive non-small-cell lung cancer in relation to p53 status.

 
On the other hand, of patients with bcl-2-negative tumors, no difference was found between survival of patients with mutations of the loop-sheet-helix motif of p53 and those with other groups of p53.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our study demonstrated that bcl-2/bax ratio status was a significant factor for a good prognosis in patients with NSCLC as described previously.26 Furthermore, mutations of the loop-sheet-helix motif of p53 were strongly correlated with overexpression of bax, whereas other mutations of p53 were correlated with lower levels of bax expression.

It becomes clear that many proteins that induce apoptosis can regulate the cell cycle.33 For example, it has been shown that expression of c-myc initiates both proliferation and apoptosis in vitro.34 In addition, expression of the tumor suppressor protein retinoblastoma (Rb), a known regulator of cell cycle progression, can repress apoptosis induced by radiation.35 The two-part mechanism of these molecules, involving both cell proliferation and apoptosis, might play an important role in the regulation of cell selection and growth arrest during differentiation into a post-mitotic state in normal organs.36

Initially, it was reported that bcl-2 and bax were regulators of apoptosis.37 Although their precise mechanisms of action remain unknown, it is widely accepted that these proteins dimerize with themselves or with each other.38,39 However, it is still unknown whether bax acts directly in their potential pathway or merely acts as an antagonist of bcl-2. Recently, many studies have demonstrated that bcl-2 and bax also have potential effects on cell proliferation.20-24 Increasing the level of bcl-2 has been shown to retard cell proliferation due to prolongation of the G1 phase of the cell cycle, sustaining the level of cyclin-dependent kinase inhibitor p27Kip1.20 In contrast, overexpression of bax has been correlated with accelerated entry into S phase, with decreased levels of p27Kip1.21 In addition, bax has been shown to reverse the inhibitory effect of bcl-2 on cell proliferation.22,23 This is likely due to neutralization of the bcl-2 activity by heterodimerization with bax as overexpression of bax alone does not accelerate cell proliferation.22 Thus, it is considered that bcl-2 might act as a direct effector in their cell proliferation pathway and that bax could act as an inhibitor of bcl-2 by combining with bcl-2.37

The observation that bcl-2 diminishes cell proliferation and that bax accelerates cell proliferation by inhibiting bcl-2 activity might reflect the biological behavior of some malignant tumors. Actually, expression of bcl-2 has been believed to be a significant factor for a good prognosis in NSCLC,26,40-43 in breast cancer,44 and in ovarian cancer.45 Previous reports using combined assessment of bcl-2 and bax expression in NSCLC demonstrated the significant difference of survival between patients with bcl-2-positive and bax-positive tumors and patients with bcl-2-negative and bax-positive tumors in stage 1 NSCLC.26 In our study of patients with NSCLC, patients with bcl-2-positive tumors had a significantly better survival than those with bcl-2-negative tumors, although bax expression alone was not related to a prognosis. In addition, of patients with bcl-2-positive tumors, the overall survival of patients with bax-positive tumors was lower than that of patients with bax-negative tumors. In contrast, of patients with bcl-2-negative tumors, patient survival was not related to bax status. Cox's regression model demonstrated that the bcl-2/bax ratio was a much stronger factor for a prognosis in NSCLC than bcl-2 status alone, which suggests that bax could act merely as an antagonist to bcl-2.

Immunohistochemical results were initially quantitated using both the HSCORE and the percentage of positive cells. Using Cox regression model analysis, the prognostic value of the bcl-2/bax ratio calculated by the percentage of positive cells demonstrated a lower Hazard ratio and lower P value compared with that of bcl-2/bax ratio calculated by HSCORE (2.924 versus 3.290, Hazard ratio; 0.0148 versus 0.0083, P value, data not shown). Therefore, the semiquantitative HSCORE method likely provides a more accurate reflection of the biological behavior of these tumors than that provided by the percentage of positive cells.

In our study, bcl-2 expression was significantly higher in squamous cell carcinomas, whereas bax expression was significantly higher in adenocarcinomas, which confirms previous observation.26 Concerning the regulation of bcl-2 and bax expressions, transcriptional dysregulation of the bcl-2 gene in the majority of non-Hodgkin's B-cell lymphomas is caused by the t(14;18) chromosomal translocation of the gene that brings it into juxtaposition with the immunoglobulin heavy-chain locus.46 However, such translocations or other gross structural alterations in the bcl-2 gene are not found in solid tumors, and the presence of unknown mechanisms of bcl-2 and bax regulation in solid tumors is still suggested.42 On the other hand, the significant difference in expression in relation to histological type of NSCLC might reflect different clinical behaviors related to histological type.47 For example, squamous cell carcinomas are likely to relapse locoregionally, whereas adenocarcinomas are likely to have distant metastasis even when the primary tumors are still small.47

Furthermore, we demonstrated that mutations of the loop-sheet-helix motif of p53 were correlated with overexpression of bax in patients with NSCLC, whereas mutations of other portions of p53 were correlated with lower levels of bax expression. In contrast, no relationship was detected between mutations of p53 and bcl-2 expression in our study. Previously, we reported that the mutation of the loop-sheet-helix motif of p53 was a significant factor for a poor prognosis in patients with NSCLC.12 The bax gene promoter region contains four motifs with homology to consensus p53-binding sites, and p53 is believed to be a direct activator of the bax gene.15 The overexpression of bax associated with mutations of the loop-sheet-helix motif of p53 might lead to a poor prognosis in patients with this subtype of bcl-2-positive tumors, by inhibiting bcl-2 activity.37

Previous studies of protein-DNA complexes have shown that {alpha}-helices are used by most of the major families of DNA-binding proteins, including the zinc finger,48 the steroid receptor,49 and the leucine zipper motif.50 p53 also uses the H2 {alpha}-helix included in the loop-sheet-helix motif, that fits in the major groove of the target DNA to make contact with the edges of the bases.11 Our results also emphasized that the H2 {alpha}-helix might play an important role in DNA recognition and that overexpression of bax might be one of the genetic factors associated with new carcinogenesis caused by mutations of the loop-sheet-helix motif of p53.

Using transforming mutants of p53, some mutations of p53 revealed enhancement of target gene expression and might therefore cause new carcinogenesis.51,52 Recently, it was reported that the codon 283-mutant p53 could cause overexpression of several target genes, including bax.53 However, there are many kinds of mutations of p53 with possible different functions.25,54 Therefore, additional investigations are required to clarify the mechanism of action of mutant p53 in relation to its target genes, including the bcl-2 family.


   Footnotes
 
Address reprint requests to Dr. Masayuki Miyake, Department of Thoracic Surgery and Department V of Oncology, Kitano Hospital, Tazuke Kofukai Medical Research Institute, 13-3, Kamiyama-cho, Kita-ku, Osaka 530-8480, Japan.

Supported in part by grants-in-aid from the Ministry of Education, Science, and Culture of Japan (10557115 and 08407040) to M. Miyake.

Accepted for publication May 11, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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