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Keywords:

  • type II alveolar pneumocytes;
  • immunohistochemistry;
  • point mutation;
  • polymerase chain reaction-single strand conformation polymorphism

Abstract

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

BACKGROUND

The ras oncogene and the p53 tumor suppressor gene play important roles in the carcinogenic process of lung carcinoma. The authors evaluated whether alterations of the ras and p53 proteins may contribute to the development of lung carcinoma in patients with idiopathic pulmonary fibrosis (IPF) and whether such alterations may explain the high incidence of lung carcinoma among patients with IPF.

METHODS

Lung tissues were obtained from 35 patients who had IPF without complications of lung carcinoma and from 36 patients who had IPF with complications of lung carcinoma. Altered expression of ras and p53 proteins was evaluated by immunohistochemistry, and mutations of both genes were evaluated by polymerase chain reaction-single strand conformation polymorphism and sequencing analyses.

RESULTS

The frequency of expression of ras protein in type II alveolar pneumocytes was significantly greater in lung tissues from patients with IPF who had lung carcinoma compared with lung tissues from patients with IPF who did not have lung carcinoma (75% vs. 40%, respectively; P < 0.01). K-ras point mutation in codon 12 (GGT to GTT transversion) was detected in lung tissue with interstitial pneumonia, in which ras protein was overexpressed in type II alveolar pneumocytes obtained from 2 of 41 patients with IPF complicated by lung carcinoma, causing amino acid substitution (Gly to Val) in both patients. A p53 mutation was detected in three of six lung tissue samples from patients who had IPF lung with positive p53 immunoreactivity, and multiple mutations were detected in two samples.

CONCLUSIONS

Expression of ras protein in type II alveolar pneumocytes and mutation in the codon 12 of K-ras gene in lung tissue may contribute to the induction of lung carcinoma in patients with IPF. Furthermore, the presence of multiple mutations in the p53 gene may explain the high incidence lung carcinoma in patients with IPF. Cancer 2002;95:624–33. © 2002 American Cancer Society.

DOI 10.1002/cncr.10708

A high incidence of complication from lung carcinoma in patients with idiopathic pulmonary fibrosis (IPF) is one of the contributing factors to a poor prognosis,1, 2 and the complications of multiple lung carcinomas are observed frequently in patients with IPF.3 A variety of mediators released from activated lymphocytes and macrophages play important roles in the pulmonary fibroproliferative response,4–6: They are associated with pulmonary fibrosis, and some of them are associated with the process of carcinogenesis. Within the reconstructed alveolar septa of a patient with IPF, proliferation and/or metaplasia of the epithelial cells are observed, and these are considered part of the precancerous state in patients with lung carcinoma.1, 7

In the carcinogenic process of lung carcinoma, point mutations and overexpression of the ras oncogene play an important role.8 It is known that overexpressed or activated and mutated ras protein continuously transmits signals to the nuclei, leading to the induction of carcinoma.9 However, the contribution of the ras oncogene to lung carcinoma complicated by IPF remains to be elucidated. In the current study, we attempted to determine whether the ras oncogene in fact does contribute to the development of lung carcinoma complicated by IPF.

The p53 tumor suppressor gene blocks the replication of damaged DNA by arresting the cell cycle in the G1 phase and preventing it from entering the S phase.10, 11 Mutation of the p53 gene (transversion from GC to TA) was observed in 40% of patients with the onset of carcinoma associated with smoking, and the mutation also was observed at a stage of mild dysplasia, which is a relatively early stage in the process of multistep carcinogenesis.12–15 Furthermore, the number of p53 positive epithelial cells increased with the severity of atypical squamous metaplasia that is regarded as a precancerous state.16, 17

We evaluated the contribution of the ras and p53 genes to the development of lung carcinoma using lung tissues obtained from patients who had IPF with and without the complications of lung carcinoma and in lung tissues obtained from patients with primary lung carcinoma. The expression of ras protein and p53 protein in individual cells was detected by immunohistochemical staining, and point mutations were observed using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis.

MATERIALS AND METHODS

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

Specimens

The diagnosis of IPF was confirmed once the following criteria were met: 1) the presence of clinical symptoms, such as dry cough and breathlessness during exertion; 2) the presence of diffusely distributed, ground glass or small, nodular opacities on a chest roentgenogram; 3) the presence of a restrictive disorder on pulmonary function tests; 4) the absence of complications from infectious diseases; and 5) the presence of histologic findings identical to those of usual interstitial pneumonia.18 In addition, patients with pulmonary fibrosis that was associated with collagen vascular disease were excluded from a diagnosis of IPF. Lung tissue specimens were obtained by open lung biopsy or by autopsy from 35 patients with IPF who did not have complications of lung carcinoma (22 men and 13 women; mean age, 66.0 years ± 1.5 years; age range, 49–92 years) and from 36 patients with IPF who had complications of lung carcinoma (27 men and 9 women; mean age, 67.6 years ± 1.4 years; age range, 49–83 years) (Tables 1 and 2). Tissue specimens were fixed immediately with formalin and embedded in paraffin. For disease control specimens, lung tissue specimens also were obtained from patients with primary lung carcinoma who were matched for age, gender, and histologic type of lung carcinoma. These lung tissue specimens were fixed similarly with formalin and embedded in paraffin. Serial 2-μm-thick sections were prepared. Hematoxylin and eosin staining was performed in the initial specimen for histologic diagnosis, and immunostaining was performed in the other specimens. Moreover, 10 μm-thick sections also were prepared for DNA extraction.

Table 1. Clinical Information on Patients with Idiopathic Pulmonary Fibrosis Complicated by Lung Cancer Carcinoma
PatientGenderAge (yr)Smoking habitS.I.Histologyrasp53
EpiGlaT-IICaMutationMetCaMutation
  1. S.I.: smoking index (pack-years); Epi: bronchial epithelial cell; Gla: bronchial gland cell; T-II: type II alveolar pneumocyte; Ca: lung carcinoma; M: male; F: female; Met: metaplasia; Adeno: adenocarcinoma; SCC: squamous cell carcinoma; Large: large cell carcinoma; NA: not applicable; Small: small cell carcinoma; Others: undifferentiated cell carcinoma; +: strong immunoreactivity (> 10%); ±: faint immunoreactivity (< 10%); −: no immunoreactivity.

1M51Yes62.0Adeno++++
2F72NoSmall±±NA
3M72Yes17.5Small±±NANA
4M49Yes77.5Large±±+NA
5F69NoLarge+±+NA
6M79Yes118.0Others+±+++NA
7F51NoAdeno±+++++
8M66Yes102.0Small+±+++NA
9M63Yes82.0Small++++NA
10M69Yes54.0Small++++
11F75NoAdeno±+
12F80NoAdeno+++
13M64Yes88.0Adeno++++NA
14F63NoOthersNA+NA
15M71Yes70.0SCC+++NA
16M68Yes68.0Large±++NA
17M68Yes87.5Small±+NANA
18M83NoAdeno+NANA
19M58Yes30.0Small+±+NA
20F76Yes60.0Adeno++NA
21F75NoOthers++NA+
22M58Yes38.0SmallNANA
23M59Yes55.0SCC++++
24M73Yes30.0Others±±+++NA
25M64Yes42.5SCCNA++NA
26M70Yes70.0SCC++++++
27M80Yes87.5SCC++++++
28M68Yes21.5Large++++++NA
29M77Yes114.0Adeno++++NA
30M64Yes88.0SCC±±++NA
31M78Yes60.0Adeno±++NA
32M66Yes92.0Adeno++++NA
33F61Yes25.0SCC+++NA
34M66Yes88.0SCC±++NA+NA
35M73Yes85.0Adeno++++NA
36M56Yes45.0Small±±++NA
Table 2. Clinical Information on Patients with Idiopathic Pulmonary Fibrosis
PatientGenderAge (yr)Smoking habitS.I.rasp53
EpiGlaT-IIMutationMetMutation
  1. S.I.: smoking index (pack-years); Epi: bronchial epithelial cell; Gla: bronchial gland cell; T-II: type II alveolar pneumocyte; Met: metaplasia; F: female; M: male; NA: not applicable; +: strong immunoreactivity (> 10%); ±: faint immunoreactivity (< 10%); −: no immunoreactivity.

37F67Yes0.3±NANA
38F67No+++NA
39M66Yes62.0+±NA+NA
40M67Yes141.0+NANA
41M62NoNA+NA
42M57Yes70.0++±NANA
43M92Yes140.0±NANA
44M67Yes125.0++
45M62Yes60.0++NANA
46M57Yes74.0+±NANA
47M57Yes75.0++NANA
48M49Yes64.0+++NA
49F60No++NANA
50F68No++NA
51F77No+NANA
52M73Yes120.0++NA
53F76NoNANA
54F67No++++NA
55F81No++NA
56F71No++++NA
57M64Yes88.0±NA+NA
58M59Yes198.0+++NA
59M59Yes111.0NANA
60M49Yes56.0+NA
61M60Yes80.0++++NA
62M58Yes37.0++±NANA
63M70Yes30.0++NA
64F69Yes26.5+NANA
65M67Yes94.0±±NA+NA
66F60No±NANA
67F71No±NA+NA
68M63No+++NA
69M73Yes106.0±±NA+
70M73Yes106.0++NA
71M72Yes50.0+NA+NA

Immunohistochemistry

Immunohistochemistry was performed on formalin fixed, paraffin embedded tissue specimens using the avidin-biotin complex method with minor modifications. The slides first were deparaffinized in graded ethanol concentrations and incubated with 0.3% hydrogen peroxide in phosphate-buffered saline for 30 minutes to block endogenous peroxidase activity. The sections were incubated overnight at 4 °C with primary antibodies against rat antihuman v-H-ras (Y13-259; Oncogene Research Products, MA) for ras protein and mouse antihuman p53 protein (DO7; DAKO A/S, Denmark) for p53 protein: Both antibodies were diluted 100 times before use. The Y13-259 antibody reportedly is able to detect human ras protein,19 and the DO7 antibody reportedly detects p53 protein.20 These primary antibodies were detected with affinity-purified, biotin-conjugated horse antirat immunoglobulin G (IgG) for ras protein and antimouse IgG for p53 protein, respectively. Subsequently, horseradish peroxidase-labeled streptavidin was applied followed by incubation with diaminobenzidine. Eight to ten sections along with the control slides were processed simultaneously. During development with diaminobenzidine, one of the pulmonary fibrosis specimens was monitored visually by light microscopy to determine the time for maximum signal-to-background generation (usually between 120 seconds and 180 seconds). The remaining tissue specimens in that batch were then developed for the same duration.

Assessment of Immunostaining Results

NIH/3T3 cells were used as a positive control of immunoreactivity for ras protein. When > 10% of the cytoplasm of individual cells or of lung carcinoma cells was stained positively by immunostaining with the v-H-ras antibody, Y13-259, it was considered positive for the ras protein. Lung adenocarcinoma tissue specimens in which p53 mutation was confirmed by PCR-SSCP analysis were used as a positive control for immunoreactivity to p53 protein. When > 10% of the nucleus of individual cells or lung carcinoma cells was stained positively stained by immunostaining with the p53 antibody, DO-7, it was considered positive for the p53 protein.

Rates of individual cells that were positive for the ras protein or the p53 protein were compared between patients with IPF and patients with IPF complicated by lung carcinoma using lung tissues with interstitial pneumonia. Moreover, the rates of lung carcinoma cells that were positive for the ras protein or the p53 protein were compared between lung tissue specimens from patients with IPF complicated by lung carcinoma and specimens from patients with primary lung carcinoma.

PCR-SSCP Analysis

Detection of mutations in codons 12, 13, and 61 of the K-ras gene and in exons 5–8 of the p53 gene were evaluated by PCR-SSCP analysis. Initially, DNA was extracted from lung tissues according to the protocol for the DNeasy Tissue Kit® (Qiagen GmbH, Germany). Briefly, 10-μm-thick, paraffin embedded sections were incubated with 1200 μL of xylene and centrifuged at 15,000 rpm for 5 minutes. The supernatant was substituted twice with 100% ethanol and was incubated at 37 °C for 15 minutes. Then, the supernatant was supplemented with 180 μL of ATL (tissue lysis) buffer and 20 μL of proteinase K and was incubated at 55 °C until the lung tissues were completely dissolved. Subsequently, 200 μL of AL (lysis) buffer were added, and the DNA solution was incubated at 70 °C for 10 minutes. After supplementing the DNA solution with 200 μL of 100% ethanol, the solution was stirred sufficiently and transferred to the DNeasy minicolumn for centrifugation at 10,000 rpm for 1 minute. Subsequently, 500 μL of AW1 (washing) buffer was added, and the solution was centrifuged at 8000 rpm for 1 minute. Then, 500 μL of AW1 buffer was added again, and the DNA solution was centrifuged at 15,000 rpm for 3 minutes. A direct application of 200 μL of AE (elation) buffer to the DNeasy membrane was followed by incubation at room temperature for 1 minute. Then, the DNeasy membrane was centrifuged at 10,000 rpm for 1 minute to elute DNA. The sequence of the PCR primer pairs were as follows; K-ras codons 12 and 13: forward, 5′-GACTGAATATAAACTTGTGG-3′; reverse, 5′-CTATTGTTGGATCATATTCG-3′; K-ras codon 16: forward, 5′-TTCCTACAGGAAGCAAGTAG-3′; reverse, 5′-CACAAAGAAAGCCCTCCCCA-3′; p53 exon 5: forward, 5′-CTCTTCCTGCAGTACTCCCCTGC-3′; reverse, 5′-GCCCAGCTGCTCACCATCGCTA-3′; p53 exon 6: forward, 5′-GATTGCTCTTAGGTCTGGCCCCTC-3′; reverse, 5′-GGCCACTGACAACCACCCTTAACC-3′; p53 exon 7: forward 5′-GTGTTGTCTCCTAGGTTGGCTCTG-3′, reverse 5′-CAAGTGGCTCCTGACCTGGAGTC-3′; and p53 exon 8: forward, 5′-CCTATCCTGAGTAGTGGTAA-3′; reverse, 5′-GTCCTGCTTGCTTACCTCGC-3′. PCR analysis was performed as follows: 30 cycles of denaturation at 95 °C for 1 minute; annealing for 1 minute at 55 °C for K-ras and at 60 °C for p53 exons 5–8; extension at 72 °C for 1 minute; and final extension at 72 °C for 7 minutes. Using 2% agarose gels, 8 μL of PCR products and 2 μL of sample buffer were electrophoresed at 100 volts for 20 minutes, and the presence of DNA samples was confirmed under ultraviolet lights. Subsequently, 6 μL of PCR products were mixed with 6 μL of SSCP degeneration reagent (95% formamide, 5% xylene cyanol solution, and 10 mg buromophenol blue), incubated at 95 °C for 5 minutes, and placed on ice. Using the Gene Gel Excel 12.5/24 Kit® (Amersham Pharmacia Biotech, Sweden), DNA samples were electrophoresed again at 600 volts for 90 minutes and then visualized using the silver-impregnation method.

DNA Sequence

The genomic PCR products, which showed abnormal bands in the SSCP analysis, were cloned with the pGEMR-T Easy Vector System II® (Promega, Madison, WI) according to the manufacturer's instructions. Twelve colonies were collected from each dish, and plasmid DNA was obtained. Direct sequencing of purified, recombinant plasmid DNA was then carried out using the didexy chain termination method using the Big Dye Terminator cycle sequencing kit (Perkin-Elmer Corporation, Foster City, CA). Cycle sequencing was performed following the manufacture's protocol. Briefly, this involved 30 cycles of denaturation at 95 °C for 20 seconds, annealing at 54 °C for 30 seconds, and extension at 72 °C for 3 minutes using the following sequencing primers: SP6 (5′-TATTTAGGTGACACTATAG-3′) and T7 (5′-TAATACGACTCACTATAGGG-3′). PCR-SSCP analysis was performed twice using proof-reading DNA polymerase, Pyrobest (TaKaRa Co., Tokyo, Japan) to rule out the PCR artifact and contamination.

Statistical Analysis

Comparisons of continuous parameters among the patients with IPF, patients with IPF complicated by lung carcinoma, and patients with primary lung carcinoma were calculated using a one-way analysis of variance. Comparisons of discontinuous parameters among the groups were calculated using chi-square tests with the Yates correction as needed. The comparison between patients with IPF and patients with IPF complicated by lung carcinoma was calculated with a Student unpaired t test. Differences were considered significant at the P < 0.05 level.

RESULTS

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

Analysis of Patients

Table 3 shows the mean ages, gender distribution, ratios of smokers, and smokers' average life-long cigarette consumption (pack-years) for the 35 patients with IPF alone and for the 36 patients with IPF complicated by lung carcinoma. There were no significant differences in the respective background factors between the two groups. Histologic types of lung carcinoma among patients with IPF complicated by lung carcinoma and patients with primary lung carcinoma are also described in Table 3. The incidence of the respective histologic subtypes of lung carcinoma did not differ between the two groups.

Table 3. Background Analysis of the Patients with Idiopathic Pulmonary Fibrosis, Idiopathic Pulmonary Fibrosis Complicated by Lung Carcinoma, and Primary Lung Carcinoma
CharacteristicIPFIPF-CALCP value
  1. IPF: idiopathic pulmonary fibrosis; IPF-CA: idiopathic pulmonary fibrosis complicated by lung carcinoma; LC: primary lung carcinoma; N.S.: not significant; SEM: standard error of the mean.

No. of patients353669
Age in yrs (mean ± SEM)66.0 ± 1.467.6 ± 1.564.6 ± 1.5N.S.
Gender (male/female)22/1327/941/28N.S.
Ratio of smokers (%)65.777.859.4N.S.
Smoking Index (pack-years)83.2 ± 9.366.4 ± 5.364.5 ± 11.1N.S.
Tumor histology: No. (%)    
 Adenocarcinoma11 (30.6)35 (50.7)N.S.
 Squamous cell carcinoma8 (22.2)15 (21.7)N.S.
 Large cell carcinoma4 (11.1)1 (1.4)N.S.
 Small cell carcinoma9 (25.0)11 (15.9)N.S.
 Undifferentiated carcinoma4 (11.1)7 (10.1)N.S.

Immunohistochemical Detection of ras Protein and p53 Protein in Lung Tissue from Patients with IPF Alone and with IPF Complicated by Lung Carcinoma

Positive immunoreactivity for the ras protein in NIH/3T3 cells is shown in Figure 1A. Positive immunoreactivity for ras protein was observed in the cytoplasm of type II alveolar pneumocytes, bronchial epithelial cells, and bronchial gland cells in lung tissue with interstitial pneumonia (Fig. 1B–D). Positive rates in type II alveolar pneumocytes were significantly greater in lungs obtained from the patients who had IPF complicated by lung carcinoma compared with the patients who had IPF without lung carcinoma: 27 of 36 patients (75%) compared with 14 of 35 patients (40%), respectively (P < 0.01) (Fig. 2). When findings in lung tissues obtained from patients with IPF alone and from patients with IPF complicated by lung carcinoma were combined for evaluation, type II alveolar pneumocytes that were positive for the ras protein were observed in 41 of 71 patients (58%). Twenty-seven of 41 patients with IPF (66%) also had complications from lung carcinoma. However, of the 30 patients with IPF who were negative for the ras protein, only 8 patients (27%) had complications from lung carcinoma. This represented a significant difference between the two groups (P < 0.01).

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Figure 1. Immunohistochemical staining for ras protein in lung tissues from patients with idiopathic pulmonary fibrosis (IPF). (A) Positive control (brown) with v-H-ras antibody (Y13-259) on NIH/3T3 cells. (B) Positive cytoplasmic immunoreactivity for ras protein on proliferated type II alveolar pneumocytes (brown) in lung tissue from a patient with IPF. (C) Positive immunoreactivity for ras protein on bronchial epithelial cells (brown) in lung tissue from a patient with IPF. (D) Positive immunoreactivity for ras protein on bronchial gland cells in lung tissue from a patient with IPF.

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Figure 2. Positive ras immunoreactivity rates in type II alveolar pneumocytes, bronchial epithelial cells, and bronchial gland cells are compared between patients with idiopathic pulmonary fibrosis (IPF) and patients with IPF complicated by lung carcinoma. The positivity rates of ras proteins in type II alveolar pneumocytes were significantly higher in lung tissues from patients with IPF complicated by lung carcinoma compared with lung tissues from patients with IPF who did not have lung carcinoma (asterisk; P < 0.01). The white square indicates the positivity rate of ras proteins in patients with IPF; and the oblique square indicates the positivity rate of ras proteins in patients with IPF complicated by lung carcinoma. Type II: type II alveolar pneumocytes; Epi: bronchial epithelial cells; Gla: bronchial gland cells.

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Positive immunoreactivity for p53 protein was observed in the nuclei of metaplastic cells. There were no significant differences in positive rates for p53 protein between lung tissue from patients with IPF alone and from patients with IPF complicated by lung carcinoma: 12 of 35 patients (34%) versus 8 of 36 patients (22%), respectively. Lung tissue from the patients with IPF complicated by lung carcinoma was classified according to the chromatic response of complicating lung carcinoma against p53 protein. Subsequently, when background factors were compared among the patients with IPF complicated by lung carcinoma, positive rates for p53 protein in lung carcinoma tissues from smokers were significantly greater compared with the positive rates for p53 protein in lung carcinoma tissues from nonsmokers: 13 of 16 patients (81%) versus 9 of 20 patients (45%), respectively (P < 0.05).

Immunohistochemical Detection of ras Protein and p53 Protein in Lung Tissue from Patients with IPF Complicated by Lung Carcinoma and from Patients with Primary Lung Carcinoma

Positive immunoreactivity for ras protein was observed in the cytoplasm of tumor cells in lung carcinoma tissue obtained from 11 of 36 patients (31%) with IPF complicated by lung carcinoma, and positive immunoreactivity for p53 protein was observed in the nuclei of tumor cells obtained from 15 of 36 patients (42%) with IPF complicated by lung carcinoma (Fig. 3). Positive immunoreactivity for ras protein was observed in the cytoplasm of tumor cells in lung carcinoma tissues obtained from 15 of 69 patients (22%) with lung carcinoma, and positive immunoreactivity for p53 protein was observed in the nuclei of tumor cells obtained from 45 of 69 patients (65%) with primary lung carcinoma. There were no significant differences in the positive rates for ras protein and p53 protein between the two groups. When both groups were classified according to the histologic types of lung carcinoma to compare positive rates for ras protein and p53 protein, no significant differences were found between the two groups. We were able to examine ras expression in normal alveolar spaces that were adjacent to tumor tissues in 61 patients with lung carcinoma (8 patients with only small tissue samples were available were omitted). Positive immunoreactivity for ras protein in alveolar pneumocytes could not be observed in any specimens.

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Figure 3. Immunohistochemical staining for p53 protein in lung tissues from patients with idiopathic pulmonary fibrosis (IPF). (A) Positive control (brown) with p53 antibody (DO7) on lung carcinoma cells. (B) Positive nuclear immunoreactivity for p53 protein on metaplasia cells (brown) in lung tissue from a patient with IPF.

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Mutation Analysis of the K-ras and p53 Genes

Using lung tissues in which ras proteins were overexpressed on type II alveolar pneumocytes, we evaluated whether there were point mutations in codons 12, 13, and 61 of the K-ras oncogene during the development of lung carcinoma in patients with IPF. Point mutation in codon 12 (GGT to GTT transversion) was detected in lung tissue with interstitial pneumonia obtained from 2 of 41 patients with IPF complicated by lung carcinoma (Patients 1 and 7), causing amino acid substitution (G to V) in both patients (Fig. 4). In addition, lung carcinoma tissues from Patients 1 and 7 were evaluated for point mutations, and no point mutations were observed in either patient.

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Figure 4. Single-strand conformation polymorphism (SSCP) and sequencing analyses of the K-ras gene (codon 12) in lung tissue from a patient with idiopathic pulmonary fibrosis (IPF). (A) Note the abnormal bands (arrows), in accordance with mutation control (P), in Patients 1 and 7 by polymerase chain reaction-SSCP analysis of the K-ras gene (codon 12). N: normal lung tissue; P: positive control with K-ras codon 12 mutation. (B) Note the G-to-T transversion with Gly to Val amino acid substitution by sequencing analysis of the K-ras gene (codon 12) in Patient 7.

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Using six tissue samples from patients with IPF in which p53 proteins were overexpressed on metaplasia cells and six tissue samples in which p53 proteins were not detected on metaplasia cells, we evaluated whether there were point mutations on exons 5–8 of the p53 gene. Although there were no mutations in six samples that were negative for p53 immunoreactivity (Patients 1, 2, 10, 11, 12, and 44), mutations were detected in three of six samples that were positive for p53 immunoreactivity (Patients 21, 26, and 27). In addition, multiple numbers of mutations were detected in samples from Patients 26 and 27 (Table 4).

Table 4. Sequencing Analysis of the p53 Gene in 12 Idiopathic Pulmonary Fibrosis Lung Tissue Samples
Patientp53 stainingp53 mutation (exon, codon, sequence, and amino acid)
1NegativeNone
2NegativeNone
7PositiveNone
10PositiveNone
11NegativeNone
12NegativeNone
21PositiveExon 5: codon 136, TGC CAA[RIGHTWARDS ARROW]TGC CCAA
23NegativeNone
26PositiveExon 5: codon 181, CGC[RIGHTWARDS ARROW]CAC (R[RIGHTWARDS ARROW]H); codon 136, TGC CAA[RIGHTWARDS ARROW]TGC CCAA
26PositiveExon 8: codon 275, TGT[RIGHTWARDS ARROW]TGA (C[RIGHTWARDS ARROW]stop); codon 277, TGT[RIGHTWARDS ARROW]GGT (C[RIGHTWARDS ARROW]G); codon 286, GAA[RIGHTWARDS ARROW]AAA (E[RIGHTWARDS ARROW]K); codon 301, CCA[RIGHTWARDS ARROW]TCA (D[RIGHTWARDS ARROW]S)
27PositiveExon 7: codon 230, ACC[RIGHTWARDS ARROW]AAC (T[RIGHTWARDS ARROW]N); codon 231, ACC[RIGHTWARDS ARROW]ATC (T[RIGHTWARDS ARROW]I); codon 246, ATG[RIGHTWARDS ARROW]ACG (M[RIGHTWARDS ARROW]T); codon 250, CCC[RIGHTWARDS ARROW]CTC (P[RIGHTWARDS ARROW]L)
44NegativeNone
69PositiveNone

DISCUSSION

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

The ras protein was overexpressed in type II alveolar pneumocytes in 41 of 71 patients with IPF, and point mutations of the K-ras gene were found in only 2 of 41 patients. In the normal lung, ras protein reportedly is expressed in the epithelial cells of the trachea and the bronchus, but not in alveolar pneumocytes.21 In lung carcinoma cells, we detected the overexpression of ras protein and mutations of the K-ras gene, which have been detected previously in different types of human carcinoma cells and is considered to be associated with the development of malignancy.22 Therefore, it has been assumed that a qualitative and quantitative evaluation of ras protein would provide results useful for the understanding of the carcinogenic processes.8

Although point mutations of the K-ras gene contribute to the development of primary lung carcinoma, no mutations were observed in the cancerous region that concomitantly developed IPF. Consequently, it was suggested that the inductive mechanism of lung carcinoma in association with IPF may differ from that of primary lung carcinoma. In the current study, positive rates in type II alveolar pneumocytes were significantly smaller in lung tissues obtained from patients with IPF without lung carcinoma compared with lung tissues obtained from patients with IPF complicated by lung carcinoma. Although the high rate (40%) of overexpression of ras protein, even in patients who had IPF without lung carcinoma, does not necessarily indicate the possible future occurrence of lung carcinoma, taken together with the roles of the ras protein in signal transduction, it does indicate that excessive signal transduction is ongoing and, thus, includes the potential for carcinogenesis. Furthermore, the expression of ras protein in type II alveolar pneumocytes was not always consistent with that observed in lung carcinoma cells from patients with concomitant IPF. This suggests that the overexpression of ras protein in type II alveolar pneumocytes contributes indirectly rather than directly to the induction of lung carcinoma. Another report indicated that the expression of epidermal growth factor receptor was increased in lung carcinoma cells that expressed the K-ras protein in mice.23 Although these events have not yet been investigated in humans, the study of differing levels of susceptibility to malignancy at both the cellular level and the individual level may help to enhance our understanding of the onset mechanism of lung carcinoma that develops as a complication along with IPF. Point mutations of the K-ras gene in type II alveolar pneumocytes in two of our patients was not correlated with mutations in lung carcinoma tissues. Because it is believed that atypical adenomatous hyperplasia already has point mutations of the K-ras gene,9 which are the basis of lung carcinoma, ras point mutations of the K-ras gene in type II alveolar pneumocytes may be unique to patients who have IPF with lung carcinoma.

One of the pathologic characteristics of lung tissue with IPF is the observation of type II alveolar pneumocytes and metaplastic cells that proliferate to cover the reconstructed alveolar spaces.1, 7 The presence of atypical epithelial cells also is detectable on occasion. The proliferation of atypical epithelial cells in the honeycombed lung may have some relation to the development of lung carcinoma.1 We investigated the contribution of the epithelial cells covering the alveolar spaces and the oncogenes and tumor suppressor genes to the pathogenesis of lung carcinoma. The results of the immunoreactivity studies of the ras and p53 proteins indicate that type II alveolar pneumocytes may participate in the development of lung carcinoma in association with IPF. Thus, the multiplication of type II alveolar pneumocytes in the reconstructed alveolar spaces may explain the greater incidence of lung carcinoma in patients with IPF.

Changes in p53 are observed frequently in patients with lung carcinoma.13 Even in a noncancerous state, such as dysplastic change, mutation of the p53 gene is detectable.24 In patients with primary lung carcinoma, the presence of dysplasia in the epithelial cells of the airway is regarded as a potentially precancerous state.14 In patients with squamous cell lung carcinoma, multiplication of the cells with the loss of one or more alleles of the p53 gene is observed in 31% of cells (including normal epithelial cells), indicating that the changes in p53 can be observed at relatively early stages of multistep carcinogenesis.25 In addition, it has been suggested that allelic deletion and/or missense mutation of the p53 oncogene in severely atypical epithelium located near lung carcinoma cells may be associated closely with smoking.13–15 In the current study, the expression of p53 protein in tissues with interstitial pneumonia from patients who had IPF complicated by lung carcinoma was not greater than the expression of p53 protein in similar tissues from patients who had IPF without lung carcinoma. Furthermore, not all of the overexpressed p53 proteins consistently showed genetic abnormalities. Positive immunoreactivity for the p53 protein in interstitial pneumonia lesions indicates the overexpression of wild type p53 protein in addition to the presence of missense mutation, which are confirmed in lung carcinoma lesions.26 Moreover, point mutation of the p53 gene was detected in three patients with IPF who were positive for p53 immunoreactivity, and two of those patients showed multiple mutations. These results indicate that chronic inflammation from IPF damages the p53 gene, resulting in the development of cloned cells with plural p53 mutations in the identical case of IPF, and that interstitial pneumonia lesions of IPF imply a precancerous status of lung carcinoma status with the risk of developing multiple tumors.

In conclusion, the expression of ras protein in type II alveolar pneumocytes with point mutation in codon 12 of the K-ras gene in lung tissue may contribute to the induction of lung carcinoma in patients with lung carcinoma accompanied by IPF. Furthermore, the presence of multiple mutations in the p53 gene may explain the high incidence of IPF complicated by lung carcinoma.

Acknowledgements

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

The authors thank Professor Takashi Yoshiki and Assistant Professor Akemi Wakisaka (First Department of Pathology, School of Medicine, Hokkaido University, Sapporo, Japan) and Dr. Hidetoshi Satoh (Division of Pathology, Sapporo City General Hospital, Sapporo, Japan) for their kind gift of lung tissues, Professor Kazuo Nagashima (Second Department of Pathology, School of Medicine, Hokkaido University, Sapporo, Japan) and Professor Yasunori Fujioka (Department of Pathology, School of Medicine, Kyorin University, Tokyo, Japan) for their kind suggestion of histologic evaluation, and Miss Nozomi Kobayashi (Department of Surgical Pathology, Hokkaido University Medical Hospital, Tokyo, Japan) for her assistance with the experiments.

REFERENCES

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