Cellular immortality: A late event in the progression of human squamous cell carcinoma of the head and neck associated with p53 alteration and a high frequency of allele loss

Abstract

Many human tumors contain variant cells that, unlike their normal counterparts, possess indefinite proliferative potential in vitro. However, little is known of the relevance of these immortal cells to human carcinomas in vivo. To investigate immortality in a human tumor system, we established cultures from different stages of head and neck squamous carcinoma (SCC-HN). All the neoplastic cultures were transformed because they showed very low cornification in surface or suspension culture and were partially or completely resistant to suspension-induced death. Immortal variants were not detected in premalignant erythroplakia cultures, but their frequency increased with tumor progression, indicating that immortality is a late event in carcinogenesis. Some late-stage carcinomas still produced senescent cultures, but, significantly, all recurrent tumors were immortal. Immortal but not senescent carcinoma cultures were associated with p53 dysfunction and a high frequency of allele loss, indicative of tumor suppressor gene inactivation. These results show that there are at least two classes of human SCC-HN that are phenotypically and genotypically distinct and that the pathological stage of a given tumor is not necessarily indicative of the kind of cells it contains.

Evidence for the inactivation of multiple replicative lifespan genes in immortal human squamous cell carcinoma keratinocytes

“…We are con®dent that the changes observed did not occur in vitro because we used a culture system where malignant keratinocytes remain phenotypically stable for more than 200 population doublings (Rheinwald and Beckett, 1981) and we always perform genetic analysis on the cultures when they have completed 30 ± 50 population doublings without any observed slowed proliferation or crisis (Loughran et al, 1994;Edington et al, 1995). Furthermore, we tested lines BICR 3 and BICR 63 for LOH at several loci on chromosomes 1, 4, 6 and 7 at 30 population doublings and again at 130 population doublings and found them still to be heterozygous (data not shown), thus illustrating the genetic stability of the lines during this period.…”

“…This suggests that the process involves one or more recessive inactivating mutations. Two candidates are the p53 and the CDKN2A/p16 ink4A genes which are both inactivated or deleted in immortal but not usually in replicatively senescent neoplastic keratinocytes (Burns et al, 1993Edington et al, 1995;Loughran et al, 1994Loughran et al, , 1996 and we have suggested that the loss of these two suppressor genes in concert is a necessary but insu cient condition to sustain the immortal keratinocyte phenotype in human SCC-HN (Loughran et al, 1994(Loughran et al, , 1996.…”

“…None of our senescent telomerase-negative cultures showed LOH at these loci (Table 2). Furthermore, culture BICR 7 which appears to be an example of a cell line in crisis (Edington et al, 1995), was telomerase-negative at early passage and retained heterozygosity at D3S1478 (Table 2, Figure 4a). Even at later passage at high density BICR 7 replicated poorly and possessed less than 20% of the telomerase activity of the immortal lines.…”

“…Squamous cell carcinoma is an excellent system in which to address this question, since normal and malignant keratinocytes can be cultured under essentially the same conditons (Rheinwald and Beckett, 1981;Edington et al, 1995) and animal models of this tumor are available (Balmain and Brown, 1988). Our previous work has shown that immortal keratinocytes arise late during the progression of squamous cell carcinoma of the head and neck (SCC-HN, Edington et al, 1995), that the immortal phenotype is recessive to the phenomenon of limited replicative lifespan and involves a high frequency of allele loss (Edington et al, 1995). This suggests that the process involves one or more recessive inactivating mutations.…”

Amplification, increased dosage and in situ expression of the telomerase RNA gene in human cancer

“…As telomerase is inactivated before or soon after birth , telomeric attrition is experienced by all normal somatic cells studied and has been suggested as a mechanism by which human cells count replication events and limit their clonal proliferation by replicative senescence (Harley et al, 1990). The presence of telomerase in immortal human cell populations and malignant tumours and its general absence prior to this stage, has led to the suggestion that cellular immortalization and telomerase reactivation are essential for the progression and clinical lethality of most human tumours, but not for their formation (Edington et al, 1995;Kim et al, 1994).…”