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TRANSFORMATION OF TRACHEAL EPITHELIAL CELLS AND THE ROLE OF TRANSFORMING GROWTH FACTOR (TGF) AND P53 IN THE LUNG C ANCER PROGRESSION Cheng, Shu-jun; Wang, Hong; Lin, Li-min; Chen, Lei; Guo, Shu-pin; Fen, Ji-nong; Han, Nai-jun; Sun, Han-xiao Cancer Institute, CAMS and PUMC, Beijing 10021, China Although lung cancer is one of the most common cancers in the world, little is known yet about genetic changes associated with its development. To facilitate the study of carcinogenesis of lung cancer, we have developed a number of experimental models including a rat tracheal epithelial (RTE) cell transformation system and two SV40 T-antigen immortalized human bronchial epithelial (HBE) cell lines. The purpose of this study was to examine the potential of a carcinogen to induce neoplastic transformation and its chemo- prevention. We investigated the role of growth factors, especially transforming growth factor (TGF), and p53 tumor suppressor genes in the lung cancer progression. 1. Carcinogen induced neoplastic transformation and its chemoprevention Cigarette smoking condensate (CSC), tobacco specific nitrosamine NNK, B(a)p, and coal tar pitch (CTP), which were considered as potential etiological factors for human lung cancer, were tested in the in vivo - in vitro RTE cell transformation

system. Carcinogens were given by intratracheal instillation, RTE cells were then isolated and examined in culture for the presence of preneoplastic variants. The results showed that CSC (table 1), NNK, B(a)p, and CTP can significantly increase transformation efficiency (TF) of RTE cells. Squamous cell carcinoma arose in nude mice after they were inoculated with the serially subcultured transformed cells. Since 6-Phenythyl isothiocyanate (PHITC) and epigallocatechin-3-gallate (EGCG) were considered as potential agents in lung cancer prevention, their effects on RTE trans- formation were tested. The results showed that PHITC (table 2) and EGCG (table 3) inhibited the RTE cell DNA alkylation and preneoplastic transformation induced by NNK or B(a)p separately, and may be useful in lung cancer chemoprevention. 2. The role of TGF-a, TGF-01 and p53 in neoplastic transformation of airway epithelium Most cancers develop in multiple stages. The RTE cell transformation system, which is suited to define multistages during transformation, is useful to explore mechanisms involved in neoplastic transformation of airway epithelium. - 2 -

By a clonal growth assay, altered responsiveness of neoplastically transformed RTE cell lines to selected growth factors was determined. The results (table 4) showed that transformed RTE cells lost their growth dependence on the addition of epidermal growth factor (EGF), but still required bovine pituitary extract (BPE) and bovine serum albumin (BSA) to be present for effective cell proliferation. Overexpression of the TFG-a protein was detected by immunocytochemistry in transformed (preneoplastic and neoplastic) RTE cell lines, SV40 T antigen immortalized HBE cell lines and non- small-cell lung cancer cell lines indicating that increased TGF-a expression is an early event in the multistage process of neoplastic transformation, and may play an important role in the lung cancer progression. EGF independence in the transformed RTE cells could conceivably be related to the overexpression of TGF-a which is known to share structural and functional homology with EGF. The colony forming efficiency (CFE) of normal primary and preneoplastic cells was inhibited to varying degrees by the conditioned medium (CM) prepared from preneoplastic and neoplastic RTE cells (table 5) . The inhibition was blocked by a TGF-Ql neutralizing antibody (table 6). In contrast, the CFE of neoplastic RTE cells was not affected by the CM (table 5). These - 3 -

data implied a paracrine role for TGF-P, in the RTE cell transformation. Southern blot analysis showed TGF-P, to be amplified in a SV40 T-antigen immortalized HBE cell line, a lung squamous carcinoma cell line, and a lung adenocarcinoma cell line. In addition, the structure of the TGF-P, gene may also be altered in a small-cell lung cancer cell line. Taken together, these data strongly suggest that TGF-P, plays an important role in the airway epithelium transformation. p53 expression was also studied in these experiments. Partial deletion of the p53 gene was found in the NNK- and MNNG-, but not B(a)p-transformed RTE cell lines, suggesting that deletion of the p53 gene is an important but not a necessary event in the RTE cell transformation. When a mutant p53 gene was transfected into rINK-treated preneoplastic RTE cells, cell transformation was observed. Transfection of a mutant, but not wild type p53 gene increased TGF-P, expression and its paracrine inhibition on normal RTE cells (table 8). Wild type p53 also repressed the proliferation of preneoplastic RTE cells (table 7). Alteration of TGF-a was not found in either the wild type p53 or the mutant p53 transfected RTE cells. It was reported that activation of oncogenes or inactivation of tumor suppressor genes were involved in the lung cancer development. In this study, we found that transforming - 4 -

growth factor, TGF-a and TGF-/3j, and p53 tumor suppressor gene play an important role in the lung carcinogenesis. Further studies will be required to define the relationships between TFG-a, TGF-Q, and p53 gene expression.

Table 1. Transformation of RTE cells by CSC CSC (mg/kg.bw) CFE% TF% 0 1.38 0.6 9 1.3.1 1.54* 17.5 1.04 2.75* *P<0.01 CFE: colony forming efficiency TF: transforming frequency Table 2. Effect of PHITC on NNK-induced RTE cell transformation Groups NNK (mg/kg.bw) PHITC (mmol) CFE% TF% control 0 0 1.578 1.16 NNK 30 0 1.492 3.60 NNK/PHITC 30 0.71 1.276 1.51* P<0.01 (compared with NNK group) Table 3. Effect of EGCG on B(a)p-induced RTE cell transformation Groups B(a)p EGCG CFE% TF% (mg/kg.bw)(mg/kg.bw) Control 0 0 1.57 0.68 B(a)p 25 0 1.46 5.23 B(a)p/EGCG 25 600 1.49 1.73* * P<0.01 (compared with B(a)p group) - 6 -

Table 4. Altered growth factor dependence of B(a)p-transformed RTE cell line SFM SFM-BPE SFM-BSA SFM-EGF SFM-B.B.E CFE% 9.4i1.2 0.95±0.02* 6.5±1.0* 10.6±1.5 0.0±0* PD/D 0.73±0.03 0.42±0.05 0.68±0.04 0.73±0.03 0.0±0 *P<0.01 (compared with SFM group) SFM: growth factors modified serum free medium BPE: bovine pituitary extract BSA: bovine serum albumin EGF: epidermal growth factor B.B.E.: BPE, BSA and EGF CFE: colony forming efficiency PD/D: population doubling/day Table 5. Effect of conditioned medium prepared from different cell lines on the CFE of primary, preneoplastic and neoplastic RTE cell lines Source of CM primary RTE CFE% NNK16 CFE% NNK47 CFE% B(a)p39 CFE% control CM 0.99±0.04 8.94±0.18 9.84±0.56 9.90±0.48 NNK15CM 0.02±0.09* 3.07±0.28* 9.46±0.14 9.43±0.09 NNK45CM 0.038±0.002* 3.66±0.08* 9.85±0.01 9.85±0.43 B(a)p37CM 0.017±0.004* 3.30±0.18* 9.83±0.26 9.75±0.38 * P<0.01 CM: conditioned medium NNK15, NNK16: NNK induced preneoplastic transformed RTE cell lines NNK45, NNK47: NNK induced neoplastic transformed RTE cell lines B(a)p37, B(a)p39: B(a)p induced neoplastic transformed RTE cell lines - 7 -

Table 6. Blocking of the inhibition of the conditioned medium from B(a)P41 on the proliferation of primary RTE cells by the TGF-01 neutralizing antibody primary RTE SFM SFM+B(a)p41CM SFM+B(a)p41CM+TGF-P1 Ab (6µg/ml) SFM+B(a)p41CM+TGF-01 Ab (154g/ml) SFM: modified serum free medium CFE% Relative CFE 2.62±0.06 100% 0.82±0.04 31.3% 1.19±0.07 45.4% 1.73±0.03 66.0% Table 7. Colony forming efficiency of wild and mutant p53 gene transfected RTE cell lines Group CFE% NNK21 6.60±0.6272 NNK21p53WT 5.44±0.4307* NNK21p53MT 7.13±0.2622 *P<0.05 (compared with NNK21 group) NNK21: NNK transformed RTE cell line NNK21p53WT: wild type p53 gene transferred NNK21 cell line NNK21p53MT: mutant type p53 gene transferred NNK21 cell line - 8 -

Table 8. Effect of CM harvested from wild or mutant p53 gene transfected RTE cell lines on the proliferation of primary RTE cells Source of CM primary RTE+CM CFE% primary RTE+CM PD/D Control 1.120±0.0794 0.69±0.04 NNK21 0.483±0.0252 0.56±0.01 NNK21p53WT 0.55±0.0608 0.60±0.03 NNK21p53MT 0.297±0.0569* 0.49±0.04 * P<0.01 (compared with NNK21 CM group)