Relationship of p53 molecular abnormalities with flow cytometry and growth factor receptor content in lung cancer

Abstract

This study attempts to clarify the oncological significance of the p53 molecular abnormalities and p53 expression in lung cancer (LC) and their relationship with flow cytometry (FC) parameters and epidermal growth factor receptor (EGFR). The study includes 65 samples taken from both LC and normal lung (NL). The p53 molecular abnormalities of exons 4–8 were studied by single strand conformation polymorphisms (SSCP) and the loss of heterozygosity (LOH) of exon 4 by the Metzler method. P53 protein was detected by Western blot. EGFR was determined by a radioligand assay using [¹²⁵I]EGF. The FC parameters S phase fraction (SPF), DNA index (D.I.), G1G0 and growth rate (G2M+SPF) were evaluated from cellular monosuspensions. The LC with SSCP p53 molecular abnormalities have a significantly higher EGFR content (P<0.001), SPF (P<0.007), D.I. (P<0.017) and a lower proportion of G1G0 cells (P<0.04) than LC with no molecular abnormalities. No relationship between p53 molecular abnormalities and tumor TN or evolutive events was found. Neither the relationship between the molecular results and p53 expression detected by Western blot nor that of the p53 expression detected by Western with FC parameters or EGFR could be shown. In NL the growth fraction cells decrease significantly (P<0.05) with the intensity of p53 expression. The lack of biological functionality of p53 with molecular abnormalities seemed to relate to fast growing LC whereas p53 expression detected by Western seemed more related to the wild type of p53.

Introduction

Lung cancer (LC) is the major cause of cancer death [1], and it has been estimated that there are 155 000 new cases per year in the USA, of which only 10% live more than 5 years. Moreover, more than 50% of resectable cancers have relapses within 5 years of surgical resection [2].

Chromosomal aberrations, amplification or hyperexpression of oncogenes of the myc or neu family [3], Ki-ras mutations [4], molecular alterations of the p53 gene, flow cytometry (FC) parameters 2, 5and growth factors, have all been implicated in LC. However, in LC a reliable biological prognostic marker, like ER in breast cancer, has not yet been found, and tumor prognosis is still based on the histopathology, tumor TNM and tumor stage. Therefore, there is a need for studies that try to clarify the biological and oncological significance of the new biological markers, which could help to establish a LC classification according to prognostic severity.

P53 is a 20 kb antiproliferative gene [6]located in 17p13.1 and composed of 11 exons [7]. The gene codes for a nuclear protein with a suppressive effect on the expression of the genes that regulate growth and cell proliferation [6]. P53 surveys the integrity of the cell genome, and in the case of DNA damage, p53 protein accumulates within the nucleus of the affected cell, leading to the arrest of the cell cycle and providing the cell with sufficient time to repair the damage [8]. Therefore, inactivation of the p53 gene by point mutation or chromosomal deletion causes genetic instability, accumulation of chromosomal aberrations and the development of malignant clones [8]. Abnormalities in the p53 gene have also been found in the Li-Fraumeni hereditary syndrome, which confers on the syndrome carriers a higher risk of developing breast, bone, brain or leukemic malignancies at early ages [9].

P53 mutations are very common in LC, and their frequency is related to the life span and cigarette smoking habits. Cigarette smoke contains organic polycyclic compounds that attack guanine bases, thus causing transversions in any one of the nine exons of the transcribed strand [10]. A specific mutation at the 249 codon produced by radon emanation has been described in uranium miners [11]. In studies carried out in LC cellular lines a good correspondence between immunoblot and immunohistochemistry results has been shown, but not all the molecular alterations are accompanied by p53 hyperexpression, for there are some that cause gene hypoexpression [12].

Immunohistochemistry 13, 14and molecular procedures 15, 16, 17have revealed p53 abnormalities in 50% of all non-small-cell LC (NSLC). However, the oncological significance of p53 abnormalities in LC is far from being established, and only in some studies has a relationship been reported with cellular ploidy [15], node involvement [17]or the advanced stage of the disease 16, 17. Nevertheless, most of the studies carried out in this field were done using immunohistochemical methods based on the hypothesis that the altered p53 has a longer half life than the wild p53, which causes its nuclear accumulation. However, this assumption contradicts certain results indicating that such methods are also able to detect the wild p53, which is suggested by the fact that p53 has been detected in the less differentiated cells of normal lung with a higher proliferative rate [18], in benign mammary tumors [19]and in normal human lymphocytes [20]. These results call into doubt the idea that the p53 detected by immunological methods (immunohistochemistry or Western blot) is restricted to abnormal p53, and it therefore seems possible that the results obtained by these methods might have a different oncological significance than those by the molecular methods.

Aneuploid tumors have been found in 76.8% of NSCLC, with an even higher rate in adenocarcinomas and advanced stage tumors [5]. Although in one study a higher overall survival among diploid LC was observed [5], the multivariate analysis used in a prospective study carried out on a large number of T1N0 NSCLC showed that FC had only moderate predictive value, inferior to that of the histopathological methods [21].

The presence of epidermal growth factor receptor (EGFR) has been observed in 75–87% of squamous carcinomas and in 70–75% of adenocarcinomas 22, 23, and an inverse relationship between tumor EGFR content and tumor differentiation has been reported 24, 25. However, in LC, unlike in breast cancer [26], EGFR was not found to be clinically significant [24].

The scarcity of studies that related p53, abnormalities or expression, with FC parameters has prompted us in the present study to clarify the relationship of p53 abnormalities and/or p53 expression with FC parameters and EGFR tumor content, histopathology and TNM tumor.