Apoptosis induction mediated by wild-type p53 adenoviral gene transfer in squamous cell carcinoma of the head and neck.

Cancer gene therapy strategies for inducing apoptosis in solid tumors may allow contemporary medicine to reassess its management of these cancers. We demonstrated previously that overexpression of the wild-type p53 gene in squamous cell carcinoma of the head and neck cell lines via adenovirus-mediated gene transfer suppressed growth both in vitro and in vivo. Here, we characterize the mechanism of the growth suppression by the exogenous p53 gene as a consequence of programmed cell death (apoptosis). One of the cell lines used in this study, Tu-138, harbors a mutated p53 gene, whereas the other cell line, MDA 686LN, possesses a wild-type p53 gene. DNA fragmentation was detected by electrophoresis in both cell lines after infection with the wild-type p53 adenovirus, Ad5CMV-p53. With the use of the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling method, 4.4% of the remaining viable Tu-138 cell population was identified as apoptotic as early as 15 h after inoculation with Ad5CMV-p53. The percentage of apoptotic cells increased to 31% at 22 h. In contrast, only 10% of the viable MDA 686LN cells (wt-p53) had undergone apoptosis 30 h after Ad5CMV-p53 infection, although the percentage of apoptotic cells rapidly increased to 60% at 48 h after infection. For in vivo analysis of apoptosis, nude mice in which squamous cell carcinoma of the head and neck cell lines had been implanted s.c. had exogenous wt-p53 transiently introduced to the tumor cells via Ad5CMV-p53 2 days later. In situ end labeling clearly illustrated apoptosis in the tumor cells. These results suggest that wt-p53 plays an important role in the induction of apoptosis in human head and neck cancer cell lines and that selective induction of apoptosis in cancer cells can be further explored as a strategy for cancer gene therapy.

Adenovirus-mediated transfer of the p53 gene produces rapid and generalized death of human glioma cells via apoptosis.

Wild-type p53 is involved in several aspects of cell cycle control and suppression of transformation, inducing either apoptosis or G1 block in cell cycle progression. Using a recombinant adenovirus containing the wild-type p53 cDNA, the biological effects of the newly expressed wild-type p53 protein were examined in six human glioma cell lines. Three cell lines (U-251 MG, U-373 MG, and A-172) expressed endogenous mutant p53, and the other three (U-87 MG, EFC-2, and D54 MG) expressed wild-type p53. The restoration of normal p53-encoded protein in the mutant cell lines induced apoptosis as assessed by morphological studies using nuclear staining, electron microscopy, and flow cytometric assays. In wild-type p53 cell lines, however, the overexpression of wild-type p53 did not result in apoptosis but inhibited cellular proliferation rather drastically and modified the neoplastic phenotype. Differential effects suggest two pathways for glioma oncogenesis and a possible therapeutic strategy.

Apoptosis induction by E2F-1 via adenoviral-mediated gene transfer results in growth suppression of head and neck squamous cell carcinoma cell lines.

E2F-1, a transcription factor by discovery, is thought to play a crucial role in regulating G1/S cell cycle progression. Its activity is modulated by complex formation with the retinoblastoma protein and related proteins. Overexpression of E2F-1 has been shown to induce apoptosis in quiescent fibroblasts. We constructed a recombinant E2F-1 adenovirus to test whether an overexpression of E2F-1 in head and neck squamous cell carcinoma cell lines would also induce apoptosis. Two cell lines, Tu-138 and Tu-167, were chosen for use in this study. Both cell lines harbor p53 mutations but express different levels of the retinoblastoma protein. Upon E2F-1 adenovirus infection, both cell lines expressed elevated levels of E2F-1 protein and then activated a pRb-chloramphenicol acetyltransferase reporter construct containing an E2F-1 binding motif. In vitro growth assay demonstrated that growth suppression by the E2F-1 protein was effective on both cell lines. Results from DNA fragmentation and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling analyses indicated apoptosis induction in cells infected with AdCMV-E2F-1. Moreover, ex vivo experiments in nude mice showed total suppression of tumor growth at sites that received cells infected AdCMV-E2F-1. An in vivo analysis of apoptosis using in situ end-labeling further demonstrated the induction of apoptosis by AdCMV-E2F-1 in tumor-bearing animals. These data indicate that overexpression of E2F-1 via an adenoviral vector suppresses in vitro and in vivo growth of head and neck squamous carcinoma cell lines through induction of apoptosis.

Genotypic analysis of flow-sorted and microdissected head and neck squamous lesions by whole-genome amplification.

To investigate the utility of primer extension preamplification (PEP) in the genetic analysis of head and neck squamous tumorigenesis, microsatellite analysis was performed on matched deoxyribonucleic acid (DNA) samples extracted from 32 flow-sorted and microdissected specimens before and after PEP. Eighteen fresh and nine archival specimens were taken from invasive carcinomas, and five specimens were obtained from microdissected archival premalignant squamous epithelial lesions. Identical microsatellite patterns were observed in 276 (87%) of the 319 paired PEP and non-PEP genotypes with sufficient DNA. Overall, 13 (4%) of the PEP and 28 (8.8%) of the non-PEP fresh tissue samples failed specific microsatellite amplification. All 14 PEP-archival specimens were successfully amplified. Sorted cells showed a higher incidence (42.8%) of loss of heterozygosity (LOH) in both PEP and non-PEP samples compared with their unsorted counterparts. The results of this study indicate that (a) PEP is a simple and reliable technique for enhancing the DNA yield from small specimens; (b) flow sorting, in certain cases, improves the interpretation of genetic results; and (c) PEP may be used to compensate for PCR failure of unamplified DNA specimens in these lesions.

Flow cytometric analysis of apoptosis and bcl-2 in human solid neoplasms.

Apoptosis (programmed cell death) and the genes regulating this process (e.g., bcl-2), have recently become a focus of interest in the study of cancer development and progression. We adapted flow cytometric techniques for measuring apoptosis and bcl-2 protein in solid tissues and concomitantly determined both parameters in 65 malignant solid neoplasms. Four different fixation methods were evaluated for bcl-2 analysis using a lymphoma cell line possessing a t(14;18), and the Daudi cell line as positive and negative controls, respectively; optimal fixation was achieved using 70% ethanol. Apoptosis was determined using the terminal deoxynucleotidyl transferase (TdT) end-labeling method of (Gorczyca et al.: Cancer Res 53:1945-1951, 1993). Treated (5Gy radiation, 4 h) and untreated portions of a murine cell line were used as positive and negative controls for apoptosis induction. For tumor specimens, bcl-2 positivity ranged from 0 to 89.5% (15.8 +/- 22.9), and apoptosis (TdT end-labeling) ranged from 0.4 to 84.3% (15.9 +/- 17.0). Generally, tumors with high bcl-2 expression (> or = 20.0%) showed significantly lower numbers of apoptotic cells than those with low (< 20.0%) bcl-2 (P = 0.05). A subset of tumors, however, exhibited low values for both parameters. We also observed that the proliferative fractions of tumors with high apoptosis (> or = 15.0%) were significantly different from those with low (< 15.0%) apoptosis (P = 0.005); higher proliferative rates were associated with high apoptosis. We conclude that optimal bcl-2 analysis is achieved using ethanol fixation and that flow cytometry provides a rapid and reliable technique for the measurement of these parameters. Concurrent analysis of these markers provides detailed information on the biological characteristics of tumor subpopulations and may assist in categorizing tumors for different management strategies.