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Keywords:

  • bronchioloalveolar carcinoma;
  • apoptosis;
  • Bcl-2;
  • Bax;
  • p53;
  • Bax inhibitor-1

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

BACKGROUND

The regulation of programmed cell death, or apoptosis, is crucial for normal development and for the maintenance of homeostasis. It has been shown that the novel antiapoptotic protein Bax inhibitor-1 (BI-1) represents a new type of regulator of cell death pathways controlled by Bcl-2 and Bax.

METHODS

Surgically resected lung specimens were obtained from 32 patients with peripheral adenocarcinomas, and BI-1 gene expression was examined and compared with expression of the p53, bcl-2 and Bax genes.

RESULTS

Fourteen of 32 tumors (43.8%) were positive for BI-1 gene expression by in situ hybridization. BI-1 gene expression in tumor specimens was significantly higher in adenocarcinomas with bronchioloalveolar carcinoma (BAC) and in adenocarcinomas of mixed subtypes with bronchioloalveolar spreading (14 of 17 tumors; 82.4%) than in carcinomas without it spreading. Patients who had BI-1-positive adenocarcinoma showed a relatively favorable prognosis compared with patients who had BI-1-negative adenocarcinoma. Eleven of 32 tumors (34.4%) were positive for the p53 protein, only 1 of 32 tumors (3.1%) was positive for the Bcl-2 protein, and 26 of 32 tumors (81.3%) were positive for the Bax protein. Protein expressions of p53, Bcl-2, and Bax, as detected by immunohistochemistry, were not associated with BI-1 gene expression.

CONCLUSIONS

BI-1 gene expression was restricted to tumor cells with lepidic growth and was a prognostic factor for peripheral-type adenocarcinoma. It is believed that BI-1 gene expression is conserved evolutionarily and may act as a key regulator of the apoptotic pathway in BAC. Cancer 2006. © 2005 American Cancer Society.

Lung carcinoma has become the biggest cause of cancer deaths in the world, including Japan and western countries. Adenocarcinoma of the lung is one of the most common histologic types of lung carcinoma, and its incidence continues to increase. Surgical resection of early-stage tumors remains the mainstay of treatment of nonsmall cell lung carcinoma (NSCLC). However, one-half of all patients who have no detectable metastasis at the time of surgery will die of their disease. Therefore, there is a need for prognostic markers to determine which patients require more aggressive follow-up.

The regulation of programmed cell death, or apoptosis, is crucial for normal development and for the maintenance of homeostasis. Uncontrollable cellular growth resulting from the acceleration of mitosis and inhibition of cell death can promote disease progression. Bcl-2 family proteins are involved centrally in the control of programmed cell death, with some inhibiting apoptosis (Bcl-2 and Bcl-XL) and others promoting apoptosis (Bax and Bak). The ability of Bcl-2 family proteins to regulate cell life and death is conserved across evolution. The association of Bcl-2 immunopositivity with the prognosis of patients with NSCLC is controversial.1, 2 It seems logical that Bcl-2 expression would be associated with a worse prognosis, because it implies an abnormal accumulation of neoplastic cells that are protected from apoptosis.

Recently, it was shown that the novel antiapoptotic protein Bax inhibitor-1 (BI-1), formerly known as testicular-enhanced gene transcript, represents a new type of regulator of cell death pathways controlled by Bcl-2 and Bax. The BI-1 gene was identified through a functional screening assay in yeast cells that were designed to select for human cDNA that could inhibit Bax-induced apoptosis.3 BI-1 can interact with Bcl-2, but not with Bax; and, when it is over-expressed in mammalian cells, BI-1 suppresses apoptosis induced by Bax, etoposide, staurosporine, and growth-factor deprivation, but not by Fas (CD95). BI-1 transcripts are isolated preferentially from tissues with high cell death rates, indicating a possible function for BI-1 in general cell survival. RNA blot analysis indicates that the BI-1 gene is expressed widely in vivo, including the heart, brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas. In general, BI-1 appears to be over-expressed in several human malignant tumors, such as prostate carcinoma,4 breast carcinoma,5 and brain tumors.6 Recent studies by in situ hybridization revealed that BI-1 gene expression is accentuated throughout the epithelium of the developing lung, where little apoptosis occurs.7 Therefore, BI-1 gene expression may be an important factor in the regulation of lung carcinoma growth. The objective of the current study was to demonstrate the expression of BI-1 in pulmonary adenocarcinoma and to examine its biologic implications.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Patients and Tissue Specimens

Materials used in this study consisted of surgically resected lung specimens from 32 patients (15 males and 17 females) with peripheral adenocarcinomas and with mediastinal and pulmonary hilar lymph node dissection. The patients underwent surgery during the period from January 1999 to February 2001 at the University Hospital of Tsukuba (Ibaraki, Japan). The patients were classified by clinical and pathologic stage according to the International System for Staging Lung Cancer.8 Preoperative clinical staging was evaluated mostly by enhanced computed tomography scans (chest, head, and abdomen) and bone scintigraphy scans. All patients underwent curative surgery and did not receive neoadjuvant radiotherapy or chemotherapy. Pathologically, 15 patients were classified with Stage I disease, 6 patients were classified with Stage II disease, and 11 patients were classified with Stage III disease. Eleven patients in the Stage III group included 7 patients with Stage IIIA tumors and 4 patients with Stage IIIB tumors. After surgery, the patients were followed in the Outpatient Department every 3 months and had enhanced computed tomography scans (chest, head, and abdomen) and bone scintigraphy scans every 6 months, even when they were asymptomatic. The surgically resected specimens were fixed routinely in 10% formalin and embedded in paraffin for histologic examination. All of the sections (3 μm thick), including the largest cut surface of the tumor, were stained with hematoxylin and eosin and elastica van Gieson and were examined by light microscopy. All tumors were classified according to the recent World Health Organization (WHO) classification of lung tumors.9, 10

In Situ Hybridization

In situ hybridization was performed with a digoxigenin (DIG)-labeled riboprobe of 399 nucleotides corresponding to the published mRNA sequence of the human BI-1 gene (nucleotide positions 114–513). For the generation of riboprobes, the BI-1 cDNA fragment was cloned into the vector pGEM-T (Promega, Mannheim, Germany). After linearization of the plasmid, DIG-labeled riboprobes were generated by in vitro transcription using SP6 and T7 RNA polymerase and the DIG RNA-labeling mixture (Roche, Mannheim, Germany) according to the manufacturer's instructions. Sections (3 μm thick) were cut from 10% formalin fixed and paraffin embedded specimens (32 tumors). The sections were deparaffinized, rehydrated, and washed with 0.1 M phosphate buffer, pH 7.4 (PB). After treatment with proteinase K, 4% paraformaldehyde, and 0.2 N HCl, the sections were acetylated with 0.25% acetic anhydride in 0.1 M triethanolamine, pH 8.0. Then, the sections were washed with PB, dehydrated in a graded ethanol series, and air dried. The hybridization mixture contained 50% deionized formamide; 10 mM Tris-HCl, pH 7.6; 200 μg/mL yeast tRNA; 1 × Denhardt solution; 10% dextran sulfate; 10% NaCl; 0.25% sodium dodecyl sulfate; 1 mM ethylene diamine tetraacetic acid, pH 8.0; and approximately 1.0 μg/mL DIG-labeled cRNA probe. Fifty microliters of the mixture were applied to each section, and the sections were covered with parafilm and hybridized in a humid chamber for 18 hours at 50 °C. The hybridization signal was detected immunohistochemically with an alkaline phosphatase-labeled, anti-DIG antibody (DakoCytomation Company Ltd., Kyoto, Japan; dilution, 1:400). For the color reaction, the slides were incubated in a solution containing nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate (both from Sigma-Aldrich Company, St. Louis, MO). The slides were washed in water, air dried, and mounted in Pristin Mount.

Immunohistochemistry

For the immunohistochemical analysis, 4 μm sections were cut from 10% formalin fixed and paraffin embedded specimens (32 tumors). The sections were deparaffinized in xylene, rehydrated in decreasing concentrations of ethanol, and immunostained by an automated method (Autostainer plus; DakoCytomation Company Ltd.). The following primary antibodies were used: anti-p53 (clone DO-7; DakoCytomation Company Ltd.; dilution, 1:100); anti-Bcl-2 (clone 124; DakoCytomation Company Ltd.; dilution, 1:100); and anti-Bax (clone N-20; Santa Cruz Biotechnology, Inc., Santa Cruz, CA; dilution, 1:100). The sections were counterstained lightly with hematoxylin. The slides were evaluated by standard light microscopy and were assessed for each antibody by two reviewers (R.T. and T.I.). Specimens were considered immunopositive when > 10% of tumor cells had clear evidence of immunostaining.

Statistical Analysis

Associations of categorical variables were evaluated with the Fisher exact test. Age was compared by using the Student t test. Survival curves were calculated by using the Kaplan–Meier method and then were compared with the generalized Wilcoxon test. All statistical calculations were performed with StatView for Windows, version 4.54 (Abacus Concepts, Berkeley, CA). Differences were considered statistically significant if the P value was < 0.05.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

BI-1 expression was detected by in situ hybridization in normal lung tissue, especially bronchial epithelium and type II pneumocytes (Fig. 1). Fourteen of 32 tumors (43.8%) showed BI-1 gene expression. Table 1 summarizes the associations between the results of in situ hybridization and the clinicopathologic features of the patients examined. The frequency of BI-1 gene expression in tumor specimens was significantly lower in heavy smokers, in male patients, and in patients who had with vascular vessel invasion, pleural invasion, and poor differentiation. It is noteworthy that BI-1 gene expression in tumor specimens was significantly higher in carcinomas with bronchioloalveolar spreading (14 of 17 tumors; 82.4%). Furthermore, BI-1 gene expression was restricted in tumor cells that showed differentiation to Type II pneumocytes or Clara cells (Fig. 2A). Although there was one bronchioloalveolar carcinoma (BAC) composed of tumor cells that showed goblet cell differentiation, the tumor cells were negative for BI-1 expression. Conversely, BI-1 gene expression absolutely was negative in carcinomas without bronchioloalveolar spreading, including the histologic subtypes of acinar carcinoma, papillary carcinoma, and solid carcinoma with mucin formation (Fig. 2B).

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Figure 1. Bax inhibitor-1 (BI-1) expression in normal lung tissue (in situ hybridization). BI-1 was positive in bronchial epithelial cells (arrows) (A) and in Type II pneumocytes (arrows) (B).

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Table 1. Bax Inhibitor-1 Gene Expression and Clinicopathologic Features in Patients with Pulmonary Adenocarcinoma
Clinicopathologic featuresAll patientsBax inhibitor-1 in ISHP value
No. positive (%)No. negative
  • ISH: in situ hybridization; smoking index: (the number of cigarettes smoked/day) × years; pathologic Stage I includes IA and IB, Stage II includes IIA and IIB, and Stage III includes IIIA and IIIB; N factor: lymph node involvement; Ly factor: lymphatic vessel invasion; V factor: vascular vessel invasion; MI: mitotic index.; BAC bronchioloalveolar carcinoma.

  • a

    The difference was statistically significant (P < 0.05).

  • b

    MI: 1 = < 1 per 10 high-power fields (HPF); 2 = 1-5 per 10 HPF; 3 = > 5 per 10 HPF.

All patients3214 (43.8)18 
Mean age (yrs)64.364.164.30.9573
Smoking index    
 ≥ 600143 (21.4)110.0356a
 < 6001811 (61.1)7 
Gender    
 Male153 (20.0)120.0155a
 Female1711 (64.7)6 
Pathologic stage    
 Stage I158 (53.3)70.5846
 Stage II62 (33.3)4 
 Stage III114 (36.4)7 
Lymph node status    
 N0168 (50.0)80.7224
 N1 and N2166 (37.5)10 
Ly factor    
 Negative128 (66.7)40.683
 Positive206 (30.0)14 
V factor    
 Negative1311 (84.6)20.0002a
 Positive193 (15.8)16 
Pleural invasion    
 Negative1711 (84.6)60.0155a
 Positive153 (15.8)12 
MIb    
 1122 (16.7)40.1535
 274 (57.1)3 
 3132 (15.4)11 
Differentiation    
 Well118 (72.7)30.0028a
 Moderately85 (62.5)3 
 Poorly131 (7.7)12 
Morphology    
 With BAC1714 (82.4)3< 0.0001a
 Without BAC150 (0.0)15 
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Figure 2. These photomicrographs illustrates the results of in situ hybridization for Bax inhibitor-1 (BI-1) and of immunohistochemistry for p53 and Bax in pulmonary adenocarcinoma. (A) Positive staining of tumor cells was observed with replacement of the bronchioloalveolar structure in the BI-1 gene analysis. BI-1 gene expression was restricted to tumor cells with lepidic growth. (B) Negative staining of tumor cells was observed with acinar and solid pattern growth in the BI-1 gene analysis. (C) Negative staining of tumor cells was observed with bronchioloalveolar spreading in the p53 protein analysis. (D) Positive staining of tumor cells was observed with replacement of bronchioloalveolar structure in the Bax protein analysis. Original magnification × 200 (all panels).

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Table 2 summarizes the associations between the protein expression of p53, Bcl-2, and Bax and the mRNA expression of BI-1. Eleven of 32 tumors (34.4%) showed positive immunoreactivity for the p53 protein (Fig. 2C). Only 1 of 32 tumors (3.1%) showed positive immunoreactivity for the Bcl-2 protein. Twenty-six of 32 tumors (81.3%) showed positive immunoreactivity for the Bax protein (Fig. 2D). Tumors that were positive for protein expression of these genes were compared with tumors that were positive for mRNA expression of the BI-1 gene. However, there were no significant correlations between p53 and BI-1, between Bcl-2 and BI-1, or between Bax and BI-1 (Table 2).

Table 2. Correlation Between the Expression of Bax Inhibitor-1 and Other Apoptosis-Related Genes
Immunologic analysisAll specimens (%)Bax inhibitor-1 in ISHP value
PositiveNegative
  1. ISH: in situ hybridization.

Total no. of patients321418 
p53 protein    
 Positive11 (34.4)56> 0.9999
 Negative21 (65.6)912
Bcl-2 protein    
 Positive1 (3.1)100.4375
 Negative31 (96.9)1318
Bax protein    
 Positive26 (81.3)1115> 0.9999
 Negative6 (18.8)33

Among the 32 patients, 21 patients still were alive after a median follow-up of 60 months. The overall estimated 3-year and 5-year survival rates were 63% and 58%, respectively. Patients who had a smoking index < 600 had a more favorable prognosis than patients who had a smoking index ≥ 600 (P = 0.012). However, no differences in the survival rate were observed between disease stages, TNM classifications, age groups, or genders. The prognostic implications of positive or negative expression of the BI-1, p53, and Bax genes also were examined. Figure 3 shows that patients who were positive for BI-1 had a fairly favorable prognosis compared with patients who were negative for BI-1 (P = 0.07). However, there was no correlation between prognosis and expression of p53 or Bax.

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Figure 3. These are the survival curves of patients with pulmonary adenocarcinomas. (A) Comparison between the patients who had tumors that were negative (n = 18 patients) and positive (n = 14 patients) for Bax inhibitor-1 (BI-1) gene expression. Patients who had positive results for BI-1 showed a favorable prognosis compared with patients who had negative results for BI-1, whereas expression of p53 (B) or Bax (C) did not influence the prognosis of the patients studied.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Pulmonary adenocarcinomas are characterized by a high degree of morphologic heterogeneity, which, in turn, implies both intratumor and intertumor diversity. According to the latest WHO classification of lung tumors,9 BAC is a noninvasive carcinoma with an extremely favorable prognosis. However, adenocarcinomas without a BAC component usually are less differentiated and show a relatively unfavorable prognosis. Consequently, it is believed that pulmonary adenocarcinomas consist of two different groups: adenocarcinoma with a BAC component and adenocarcinoma without a BAC component.11 In our study, all of the carcinomas with a BAC component (17 of 32 tumors) that were diagnosed as adenocarcinoma with mixed subtypes in the WHO classification were positive for BI-1 gene expression, but absolutely no expression was detected in the other histologic subtypes (such as papillary, and/or acinar, and/or solid tumors). Thus, we found that BI-1 gene expression is associated closely with morphologic differentiation to BAC.

Clinicopathologically, positive BI-1 gene expression status was associated significantly with female patients, low smoking index, patients without vascular vessel invasion or pleural invasion, and well differentiated tumors. These clinicopathologic characteristics of BI-1 gene expression appear to result from its association with BAC, because similar general features of BAC have been reported previously.11 Patients who had BI-1 gene expression had a more favorable prognosis than patients who had no expression of BI-1. This trend in prognosis also was a result of the better outcome of patients with BAC. Thus, the BI-1 gene is expressed in accordance with the cell type, suggesting that the other histologic subtypes without a BAC component lack the ability to express the BI-1 gene. Conversely, abnormal expression of p53 or Bax showed no association with prognosis in this study, which is inconsistent with the many previous reports indicating that abnormal p53 expression is correlated with an unfavorable prognosis. This discrepancy may be a result of the limited number of specimens that we examined.

Jean et al. speculated that the BI-1 gene may play a key role in lung cell survival at birth.7 Their study, which also included in situ hybridization analysis, showed marked BI-1 gene expression throughout the epithelium of the developing lung, where little apoptosis occurs. Those authors suggested that further study of BI-1 gene expression in these cells may provide new insights into the biology of lung epithelial cells during the perinatal period and potentially during repair after lung injury and lung carcinogenesis. In the current study, BI-1 gene expression was detected in the epithelial cells of normal lung tissue, suggesting that BI-1 may have an important role in the survival of epithelial cells in the normal adult lung. High levels of expression of the BI-1 gene may provide protection against apoptosis induced by some types of stimuli in tumor cells with lepidic growth and in epithelial cells of normal lung tissue. BAC or BAC component generally does not contain apoptotic tumor cells or necrosis, possibly indicating that BI-1 contributes functionally to survival of the tumor cells of BAC.

In conclusion, the current results showed that the BI-1 gene is expressed frequently in the BAC component of adenocarcinoma but is not expressed in the other histologic subtypes of pulmonary adenocarcinoma. Although the numbers of patients analyzed in this study were small, and this was a preliminary report, we speculate that the BI-1 gene may be conserved evolutionarily and may act as a key regulator of apoptosis in BAC-type adenocarcinoma. To elucidate the role of the BI-1 gene in the carcinogenesis and malignant progression of BAC-type adenocarcinoma, a larger scale analysis should be performed.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES