PUMA in head and neck cancer
Introduction
The p53 tumor suppressor is a sequence-specific transcription factor whose loss contributes to carcinogenesis [1], [2]. Functional inactivation of p53 is a very common event in many types of human cancer [3]. However, it is estimated that 50% of all cancers do not have a p53 mutation [4]. The p53 protein is postulated to function in the maintenance of genomic stability by inhibiting replication or initiating apoptosis after DNA damage [5] and p53 transactivation regulates an increasing number of downstream target gene [6]. The spectrum of genes activated (and repressed) by p53 is almost certainly influenced by many factors, including cell type [7]. The function of these gene products is diverse and already includes proteins involved in cell cycle regulation and apoptosis. Given the role of p53 as a key tumor suppressor, a continuing challenge is to completely define the set of p53 regulated genes that mediate its tumor suppressor activity. Elimination or functional inactivation of other participants of the p53-dependent pathway [8], [9], [10], [11] which mediate G1 arrest after ionizing radiation and other DNA-damaging agents [11], [12], [13] could also mimic altered or lost p53 function and contribute to malignant progression.
Many studies have been performed to identify genes that are regulated by p53 and lead to apoptosis [14]. Among these candidates, those that encode mitochondrial proteins are particularly attractive because p53-initiated apoptosis appears to proceed through a mitochondrial pathway. To date, three (Bax, Noxa and p53AIP1) p53-regulated genes that encode proteins residing in the mitochondria were identified [15], [16], [17]. Recently, a novel gene was added to this group of genes called PUMA. PUMA is remarkably conserved across species including humans and mice [18]. Moreover, deletion of chromosomal arm 19q has been reported in HNSCCs (head and neck squamous cell carcinoma) and lung cancer [19], [20], [21], [22], [23], [24]. Overexpression of PUMA can induce apoptosis regardless of the p53 genotype of the cell and induction of endogenous p53 through treatment with DNA damaging agents induces PUMA [18]. We hypothesized that PUMA mutations may, in some settings, substitute for p53 mutations in human oncogenesis. For example, in the presence of wild type p53, loss of function (by bi-allelic inactivation) could give a similar cell phenotype to p53 inactivation. Consequently we examined the role of forced expression of PUMA followed by complete sequence analysis in 30 primary human tumors (15 head/neck, and 15 lung) and panel of 10 human head/neck cancer cell lines with known p53 status (wild type or mutant).
Section snippets
Sample collection
Primary tumor and blood were collected from 30 patients undergoing surgical resection of head/neck and lung cancer. Lymphocytes were collected from blood and used a source of normal DNA. Tumor samples were promptly frozen at −80 °C after initial gross pathological examination.
Portions of the primary tumor were cut into 7 μm sections, stained with hematoxylin and eosin, and examined by light microscopy. Additional 12 μm sections were cut and placed in a mixture of 1% SDS and proteinase K at 48
Results and discussion
To determine the effect of PUMA expression in head and neck cancer we transfected an expression vector containing PUMA into two different head and neck cancer cell lines. Following selection, there was a drastic reduction in colony formation after transfection with the PUMA expression vector compared to the empty vector or to an analogous vector encoding PUMA without its BH3 domain (Fig. 1). This colony suppression was observed regardless of the p53 genotype of these isogeneic cells (WT type in
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