American Journal of Pathology, Vol. 139, No. 4, October 1991 Copyright © American Association of Pathologist

Detection of p53 in Primary Lung Tumors and Nonsmall Cell Lung Carcinoma Cell Lines J. Caamano, B. Ruggeri, S. Momiki, A. Sickler, S.Y. Zhang, and A.J.P. Klein-Szanto From the Department of Pathology, Fox Chase Cancer Center, Philadelphia, Pennsylvania

Immunohistochemical analysis of p53, a nuclear protein involved in the development of numerous human tumors, was performed in a series of50 primary nonsmall cell lung carcinomas and in a group of eight lung carcinoma cell lines. Using two mouse monoclonal antibodies, PAb18OJ and PAb421, sixteen of thirty-five (45.7%) lung adenocarcinomas and seven offifteen (46 6%) squamous cell carcinomas showed marked-to-moderate immunoreactivity. In fifty-six percent of the positive tumors more than 40% of all cells were p53 positive, and in only 17% ofpositive tumors the percentage of immunostained cells was less than ten. Although the number ofp53 negative adenocarcinomas without metastasis was larger than the number ofp53 positive tumors without metastasis, there were not clear differences between p53 positive and negative tumors with metastasis. Furthermore, six adenocarcinomas that infiltrated the pleura and/or the thoracic wall were p53 positive, whereas only two of these invasive tumors were p53 negative. From eight cell lines studied six were positive for p53. A good correlation between immunocytochemistry and immunoprecipitation was observed. Two tumorigenic and metastatic cell lines, Calu 1 and Calu 6, that were not immunoreactive also showed lack of protein by immunoprecipitation, as well as absence of mRNA in Northern analysis. In addition, Calu I showed an important gene deletion These observations point to the fact that deletions and alterations in transctiption of the p53 gene could coincide with or eventuate in an advanced malignant phenotype that nevertheless results in a p53 negative immunostain. Although this type of change cannot be detected immunohistochemically in prmary tumors without further molecular analysis the results presented herein indicate

that p53 can be detected immunohistochemically in a majority of lung tumors and that there is a tendency for more advanced adenocarcinoma stages to

exhibit positive p53 immunostain. (Am J Pathol 1991, 139:839-845)

Current evidence indicates that antioncogene alterations, especially of the p53 and to a lesser extent of the retinoblastoma gene, are a frequent occurrence in all types of lung cancer.1-5 In addition, the p53 gene located in the short arm of chromosome 17, has been reported to be a frequent site for abnormalities in several human tumors and tumor cell lines derived from neurofibrosarcomas, lymphomas, leukemias, osteogenic sarcomas, and colorectal tumors.6 The development of monoclonal antibodies against different epitopes of the p53 product7-9 has made possible its immunohistochemical detection in cell and tissue preparations. Although some of these antibodies, e.g., PAb240 is specific for the mutated form of p53, other widely used antibodies such as PAbl 801 and PAb421 detect both the mutated and the normal p53 proteins. Nevertheless, and probably because of the short half-life of the normal product, only the mutated form seems to be detectable immunohistochemically with these antibodies.1-o1 Increased expression of p53 has been demonstrated immunohistochemically in frozen sections of lung, breast, and colon tumors3 12-13 as well as in paraffin-embedded colorectal tumor fixed with periodate lysine paraformaldehyde dichromate.11 These studies on colon tumors confirmed earlier molecular evidence indicating that p53 alterations are associated with malignant progression.15 Iggo et al.3 demonstrated p53 in a large percentage of lung carcinomas using the mutation-specific antibody PAb240.9 In the present study, we compared the immuSupported by grants CA-44981 and CA-53713 from the National Institutes of Health. Accepted for publication June 12, 1991. Address reprint requests to Dr. A.J.P. Klein-Szanto, Department of Pathology, Fox Chase Cancer Center, 7701 Burholme Avenue, Philadelphia, PA 1911 1.

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noreactivity of a series of primary lung neoplasms and carcinoma cell lines with the monoclonal antibodies PAbl 801 and PAb421 that have been used to detect p53 in other tumors.11-13 In addition, we investigated biological or prognostic correlates that would emphasize the importance of the immunohistochemical detection of p53 in tissue sections and cell lines and could eventually help in understanding the role of p53 in lung-tumor development and progression.

Material and Methods Primary Lung Neoplasms A total of 50 nonsmall cell lung tumors obtained during surgical resection were snap-frozen in liquid nitrogen and stored at -70°C in optimal cutting temperature compound (OCT, Miles Inc., Elkhart, IN). The available tumors were 35 adenocarcinomas and 15 squamous cell carcinomas. Adjacent nonfrozen blocks fixed in formalin, embedded in paraffin, and stained with hematoxylin-eosin were used to establish the histopathologic diagnosis.

Cell Lines Seven human malignant epithelial cell lines derived from lung tumors were used: A427, A549, Calu-3, and SKLU-1 (adenocarcinomas), Calu-1, HU281 and SK-MES (squamous cell carcinomas), and Calu-6 (anaplastic carcinoma). Except for HU281 that was developed in our laboratory, all cells were obtained from the American Type Tissue Culture Collection. The cells were maintained under an atmosphere of 5% CO2 in antibiotic-free MEM supplemented with 10% FBS. Normal human bronchial epithelial cells (HU240) derived from an adult donor were maintained as primary cultures using previously described techniques.14

vein. After 8 weeks, or before if the animals became sick, all animals were killed, and the metastatic nodules on the surface of the lung were counted.

Southern and Northern Blot Analysis DNA was isolated from the tumor cell lines using the proteinase K/phenol extraction method.16 DNA digested with Eco RI, Hind 111, Xbal, Bam HI, was separated by electrophoresis in 0.9% agarose. Messenger RNA was isolated using the Fast Track kit (Stratagene) and separated by electrophoresis in formaldehyde/agarose gels. Both RNA and DNA were transferred to nylon filters and hybridized to random primer P32-labeled probes.17 The hybridization probe was 2-kb full-length human p53 cDNA insert from plasmid pHp53B.18

Immunocytochemistry Frozen section and cell lines fixed in acetone 40C for 15 minutes were incubated overnight at 40C with PAb1801 (dilution 1/100), PAb421 (dilution 1/30), and PAb240 (dilution 1/1 0, 1/20, and 1/50) (Oncogene Sciences, Manhasset, NY). The immunostain was performed using a biotinylated antimouse antibody kit (Vectastain, Vector, Burlingame, CA) and diaminobenzidine (1 mg/ml) with 0.02% of hydrogen peroxide. Positive controls from two known positive cases of colon carcinoma were used in each batch. All sections and cell lines were also stained without primary antibody to monitor background staining. Light hematoxylin counterstain was used in some cases, especially for illustration purposes. Percentage of positively stained nuclei were calculated counting 500 to 1000 nuclei in each slide without counterstaining. Total proteins were extracted from a dozen selected tumors, and immunoblots were prepared as described by others to confirm the presence of p53.10

Tumorigenicity, Spontaneous Metastasis, and Experimental Lung Colonization Assays

Cell Labelling and Immunoprecipitation Studies

Semiconfluent grown tumor cells were harvested by 0.05% trypsin treatment and 2 x 106 cells in 0.1 ml were injected subcutaneously in the back of 4- to 6-week-old nude mice of Balb/c background. When tumors were observed, tumor size was measured twice a week. Eight to 16 weeks later, all mice were killed when tumors reached 20 mm in diameter. The lung metastases were assessed macroscopically and by histologic examination. Experimental lung colonization assay was done by injecting IV 5 x 105 cells in 0.1 ml of tissue culture medium in the tail

Cell lines were maintained in minimum essential medium (MEM) supplemented with 10% fetal bovine serum (FBS) and incubated at 370C in a humidified 5% CO2 atmosphere. Subconfluent cell cultures were labelled for 1.5 hours with 300 microcuries of [3S]-L-methionine (ICN) in 3 ml of methionine-free MEM containing 2% dialyzed FBS at 370C after a 1-hour methionine starvation.19 Cells were washed twice in phosphate buffered saline (PBS) and extracted on ice in 1 ml of lysis buffer. Cell lysates were precleared with 50% protein A Sepharose and equivalent

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amounts of radiolabelled proteins were immunoprecipitated overnight. The following monoclonal antibodies were employed for immunoprecipitation studies: pAB 4217 directed against carboxyl-terminal amino acids 370-386 of mammalian p53 and pAB 18018 directed against amino-terminal residues 32-79 of human p53. Immune complexes were pelleted by centrifugation and washed as described.20 Pellets were resuspended in SDS-polyacrylamide electrophoresis gel (PAG) sample buffer, boiled several minutes, and applied to a 10% PAG. Gels were fixed and processed for fluorography in 20% 2.5-diphenyloxazole in dimethylsulfoxide and autoradiographed at - 700C.

Table 2. Number ofp53-positive Lung Tumors Showing Variable Percentage of Reactive Cells Percent of nuclei* stained PAbl 801 PAb421 1-10% 11-40% 41-80% 81-100%

4 6 10 3

4 6 11 2

* A total of 500-1000 cells were counted per slide in randomly selected fields.

chest wall was three times higher than the p53 negative invasive tumors. Immunoblots from selected tumors showed a good correlation with the immunohistochemical reactions (data not shown).

Results Primary Nonsmall Cell Lung Carcinomas

Lung Tumor Cell Lines

The immunohistochemical study of 50 primary lung tumors showed that 46.6% of these tumors stained positively for p53 (Table 1). Antibodies PAbl 801 and PAb421 gave similar results, i.e., intense to moderate nuclear stain in most tumors. PAb240 gave negative results with all dilutions used. Table 2 shows the percentage of cells stained with PAb1801 and PAb421, respectively. Only 17% of tumors exhibited less than 10% of immunoreactive cells, most neoplasms (56%) showed nuclear stain in more than 40% of the tumor cells although only a few (12%) exhibited p53 positive reaction in all tumor cell nuclei. The same patterns of reactivity were seen in adenocarcinomas and squamous cell carcinomas (Figure 1 A, B). A slightly higher percentage of metastatic tumors were p53 positive; nevertheless, the difference with respect to the nonmetastatic tumors was not statistically significant. Although not statistically significant, the number of p53 positive tumors invading the pleura and the

Biological Behavior of Lung Tumor Cell Lines

Table 1. Immunostain of Primary Nonsmall Cell Lung Carcinomas Tumors

p53 Positive*

p53 Negative

16

19

6 9

2 5t 14

7

8

0 4 4

0 3 5

Adenocarcinomas Total With invasion to pleura and/or thoracic wall (T3) With metastases (Ni, N2, MO) Without metastases (NO, MO) Squamous Cell Carcinomas Total With invasion to pleura and/or thoracic wall (T3) With metastasis (Ni, N2) Without metastases (NO, MO) *

7

Immunostained with PAb1801 and PAb421.

t One case without lymph-node metastases, but with a solitary

brain metastasis, was included in this group.

Except for the cell lines SKMES and SK-LU-1, the remaining six cell lines were tumorigenic when injected subcutaneously into nude mice (Table 3). The subcutaneous tumor incidence was 70 to 100% in all cell lines studied except for A 427 cells in which the incidence was lower (25%) (Table 3). The spontaneous metastatic ability of these cells, as measured by the presence of lung metastases in the sc. injected animals, showed some differences. For example, Calu 6 was highly metastatic with more than half of the animals exhibiting lung metastases, whereas A 549 was moderately metastatic with one metastatic tumor out of four. The remaining tumorigenic cell lines tested did not produce any spontaneous metastasis. Lung colonization after IV injection of the human cell lines showed similar trends, i.e., Calu 6 exhibited a high lung-colonizing ability, whereas Calu 1 and A 549 cells showed an intermediate lung-colonizing ability. Except for Calu 1, the cell lines that had no spontaneous metastatic ability also lacked the capacity to produce lung colonies after IV inoculation (Table 3).

Immunohistochemistry of Nonsmall Cell Lung Carcinoma Cell Lines Six of the eight cell lines studied showed immunoreactivity with PAb1801 and PAb421 (Table 3). Only cell line HU281 showed intense immunostain in all cells of subconfluent cultures (Figure 1 C), whereas Calu 3, A427, A549, and SKMES cells showed moderate stain in less than 5% of the cells (Figure 1 D). SKLU-1 cells showed marginal positivity in subconfluent cultures. Interestingly,

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Figure 1. Immunobistochemnical deletion ofp53 using pAB1081 as primary antibody sbows a large number of immunoreactive cells in a lung adenocarcinoma (A) and in a lung squamous cell carcinoma (B). Note that the stromal cells are not immunoreactive. Counterstained with hematoxylin, x200. Immunocytochemical demonstration ofp53 using PAb421. All HU281 cells are heavily stained (C) x400, whereas only a few A427 cells exhibit prominent nuclear stain (D), x200.

this percentage was even lower in confluent cultures. Calu 1 and Calu 6 cells did not show immunostain.

Southern and Northern Analysis of Tumor Cell Lines

Immunoprecipitation of Tumor Cell Lines

Southern blot analysis revealed a gross deletion in the p53 gene of Calu 1 cells. This cell line had a homozygous deletion that mapped 3' from the Xbal site in intron 1. Digest of Calu 1 DNA with Xbal (Figure 3A) or Eco RI (data not shown) revealed fragments of 7.5 kb and 3 kb, respectively, corresponding to the 5' region of p53 gene. The rest of the cell lines showed a normal pattern. These results were confirmed by restriction digests with Hind Ill and Bam HI (data not shown). Poly A RNAs from the eight

As shown in Figure 2, normal size p53 protein was detected in six of the eight cell lines using antibodies PAbl 801 and PAb421. Although the differences in intensity were not as obvious as with immunocytochemistry, reaction patterns were similar. As previously shown with immunocytochemistry, p53 protein was not detected in cell lysates from Calu 1 and Calu 6.

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Table 3. Tumorigenicity, Spontaneous Experimental Metastatic Potential and p53 Immunostaining of Huuman Lung Tumor Cell Lines

Histological type of original tumor

Cell lines Calu-6 A549 Calu-1 Calu-3 HU-281 A427 SKMES SK-LU-1

*Tumorigenicity

Anaplastic

5/5 4/4 7/10 5/5 5/5 1/4 0/10 0/10

Adenocarcinoma

Squamous Adenocarcinoma Squamous carcinoma Adenocarcinoma Squamous Adenocarcinoma

tSpontaneous lung metastasis 3/5

tExperimental lung colonization 5/5

1/4 0/5 0/5 0/0 0/1

2/5 1/10 0/5 0/0 0/4 0/8 0/10

Percent of p53( +) cells1 pAB421 0

pABl 801 0 1 0 3 100 1 2 0

2 0 4 100 6 1 1

*(2 x 106 cells were injected sc), no. of tumor bearing mice/no. of mice sc injected at 8 to 16 weeks. t No. of mice with spontaneous lung metastasis/no. of tumor bearing mice at 8 to 16 weeks. t (5 x 105 cell were injected iv), no. of mice with lung nodules/no.of mice treated iv at 8 weeks. 1A total of 500 to 1000 cells were counted in two different slides containing 50-70% confluent monolayer cultures.

lung carcinoma lines were extracted and analyzed by Northern blotting; p53 mRNA was not detectable in Calu 1 and a greatly reduced expression in Calu 6 cells was noted (Figure 3B).

Discussion The present immunohistochemical study of p53 in a series of primary nonsmall cell lung carcinomas and a group of cell lines derived from similar lung tumors showed that p53 can be visualized in approximately 50% of tumors. A similar percentage of colon and oral cavity carcinomas were described as positive by other investigators when either PAb1801 or PAb421 were used.10'11 1321 22 In addition, Chiba et al. described that in 45% of nonsmall cell lung carcinomas, p53 muta-

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tions were detected using an RNAse protection assay and cDNA sequencing. A slightly smaller percentage of breast cancers were p53 positive with these antibodies.12 Interestingly, Iggo et al.3 found that 70% of 31 nonsmall cell lung carcinomas were positive when using hybridoma supernatant of PAb240,2 a mutant-specific antip53 antibody. Using the commercial counterpart of this antibody, we were unable to detect p53. Nevertheless in our series of 50 lung tumors, 23 were positive with antibodies PAbl 801 and PAb421 that recognize both the wild type and the mutant p53 product. Normal tissues in the same sections do not stain, indicating that the more stable mutant p53 product in the positive tumor cells is the only p53 type detectable with these antibodies. Similar observations have been made by other investigators.1013 Conversely some p53 tumors could exhibit

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Figure 2. Immunoprecipitation ofp53 protein from [35S]-Methionine-labeled human lung cancer cell lines. Equivalent amount of radioactivity (1 x 109 cpm) were subjected to immunoprecipitation with PAb 421 (a) and PAb 1801 (b). HU240 cells arefrom aprimary human bronchial epithelial culture and served as a control. Molecular size markers are indicated (in kilodaltons).

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negative immunoreactivity, not only because of a lack of abnormalities in p53, but because of important alterations in this locus that could result from a deletion of both alleles or on alteration in transcription. This type of change has been described in some lung tumor cell lines1' 23 and was also seen in two lung cell lines described in the present report. Calu 1 cells showed an important deletion of the p53 gene together with absent expression of p53 mRNA and protein. Calu 6, although showing no gene alteration in Southern analysis, exhibited practically no mRNA or p53 protein. Both cell lines had negative immunostaining, thus pointing to the fact that negative immunoreactivity does not necessarily imply normal p53 status. No correlation could be found between the ability of primary lung tumors to metastasize and the degree of

F-

i Figure 3. Molecular analysis of nucleic acids. Southern blot analysis of p53 gene in nonsmnall cell lung carcinoma cell lines (A). Ten jig of genomic DNA of each cell line was digested with XbaI and electrophoresed in 0.9% agarose and hybridized with 2Kb Bam Hl fragment of the pHp53B probe. Northern blot analysis of p53 transcripts in the nonsmall cell lung carcinoma cell lines (B). A faint band corresponding to p53 mRNA in Calu 6 was seen after long exposure. Rehybridization with actin is sbown at the bottom of the figure.

immunostaining, and although we identified an important proportion of p53 positive tumors that invaded the thoracic wall and/or the pleura, the small number of tumors in this category precludes a definitive conclusion. A lack of a clear correlation between metastatic ability and immunoreactivity was seen in the group of eight nonsmall cell carcinoma cell lines. The two most reactive cell lines were nonmetastatic, whereas the metastatic cell line A549 was marginally p53 positive. The two other metastatic cell lines Calu 1 and Calu 6 were not immunoreactive. As already pointed out, these cell lines had important alterations in the p53 locus and expression of mRNA, indicating that eventually the total lack of p53 protein could coincide with increased metastatic behavior. It is also interesting to mention that, although all other cell lines had point mutations in the p53 gene as demon-

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strated by either binding to heat-shock protein, half-life studies, or sequencing (Caamano et al.24), not all these cell lines have the same immunohistochemical properties. Certain mutations may be more easily detectable than other mutations with the currently available antibodies. Expression of p53 has been identified as occurring during the conversion of a benign to a malignant tumor in the colon.15 The exact chronology of p53 abnormalities in lung tumors is more elusive, because of the lack of abundance of early tumors or premalignant lesions. Furthermore, the findings of Chiba et al.4 in 51 cases of nonsmall cell lung carcinoma suggest that the occurrence of p53 mutations in early and late stage tumors is similar and that p53 probably does not play an important role during tumor progression. Nevertheless, the relatively small number of cases of advanced tumors (T3 stage) in that report (n = 5) and in ours (n = 8) should introduce a note of caution before ruling out the possible role of p53 during late lung tumor progression. Future studies in larger series of early and late tumors using more sensitive reagents and techniques could improve our understanding of the role of p53 in lung carcinogenesis.

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sudomi T, Buchhagen DL, Carbone D, Piantadosi S, Koga H, Reissman PT, Slamon DJ, Holmes EC, Minna JD: Mutations in the p53 gene are frequent in primary, resected nonsmall cell lung cancer. Oncogene 1990, 5:1603-1610 5. Harbour JW, Lai S-L, Whang-Peng J, Gazdar AF, Minna JD, Kaye FJ: Abnormalities in structure and expression of the human retinoblastoma gene in SCLC. Science 1988,

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Genes Develop 1990, 4:1-8 7. Harlow E, Crawford LV, Pim DC, Williamson NM: Monoclonal antibodies specific for simian virus 40 tumor antigens. J Virol 1981, 39:861-869 8. Banks L, Matlashewski G, Crawford L: Isolation of humanp53-specific monoclonal antibodies and their use in the

studies of human p53 expression. Eur J Biochem 1986, 159:529-534 9. Gannon JV, Greaves R, Iggo R, Lane DP: Activating mutations in p53 produce a common conformational effect. A monoclonal antibody specific for the mutant form. EMBO J 1990, 5:1595-1602 10. Rodriguez NR, Rowan A, Smith MEF, Kerr IB, Bodmer WF, Gannon JV, Lane DP: p53 mutations in colorectal cancer. Proc Natl Acad Sci 1990, 87:7555-7559 11. Purdie CA, O'Grady J, Piris J, Wyllie AH, Bird CC: p53 expression in colorectal tumors. Am J Pathol 1991, 138:807813 12. Cattoretti G, Rilke F, Andreola S, D'Amato L, Delia D: p53 expression in breast cancer. Intl J Cancer 1988, 41:178183 13. Van den Berg FM, Tigges AJ, Schipper MEI, Den HartogJager FCA, Kroes WGM, Walboomers JMM: Expression of the nuclear oncogene p53 in colon tumours. J Pathol 1989, 157:193-199 14. Ura H, Bonfil RD, Reich R, Reddel R, Pfeifer A, Harris CC, Klein-Szanto AJP: Expression of type IV collagenase and procollagen genes and its correlation with the tumorigenic, invasive, and metastatic abilities of oncogene-transformed human bronchial epithelial cells. Cancer Res 1989, 49:4615-4621 15. Fearon ER, Vogelstein B: A genetic model for colorectal tumorigenesis. Cell 1990, 61:759-767 16. Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning. 2nd Edition. Cold Spring Harbor Lab Press, 1989, 9.16-9.19 17. Feinberg AP, Vogelstein B: A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 1984, 137:266-267 18. Zakut-Houri R, Bienz Tamor B, Givol D, Oren: Human p53 cellular tumor antigen: cDNA sequence and expression in COS cells. Embo J 1985, 4:1251-1255 19. Finlay CA, Hinds PW, Levine AJ: The p53 proto-oncogene can act as a suppressor of transformation. Cell 1989, 57:1083-1093 20. Hinds PW, Finlay CA, Frey AB, Levine AJ: Immunological evidence for the association of p53 with a heat shock protein, hsc70, in p53-plus-ras-transformed cell lines. Mol Cell Biol 1987, 7:2863-2869 21. Coltrera R, Zarbo R, Sakr WA, Gown AM: Suprabasal expression of PCNA/cyclin and not cytokeratin 19 is a marker of premalignancy in the oral cavity. Lab Invest 1991, 64:63A 22. Listrom MB, Fenoglio-Preiser CM: Immunochemical analysis of p53 in colon cancer. Lab Invest 1991, 64:38A 23. Takahashi T, D'Amico D, Chiba I, Buctuhagen DL, Minna JD: Identification of intronic point mutations as an alternative mechanism for p53 inactivation in lung cancer. J Clin Invest 1990, 86:363-369 24. Lehman TA, Bennett W, Metcalf RA, Welsh J, Ecker J, Modali R, Ullrich S, Romano JW, Appella E, Testa J, Gerwin BI, Harris CC: p53 mutations, ras mutations and p53-heat shock 70 protein complexes in human lung carcinoma cell lines. Cancer Res 1991, 51:4090-4096