A p53 dose-response relationship for sensitivity to DNA damage in isogenic teratocarcinoma cells.

Teratocarcinomas are tumors that arise from primordial germ cells and are readily curable with DNA-damaging chemotherapeutic drugs. Teratocarcinoma cells ex vivo in tissue culture are also relatively chemosensitive and undergo apoptotic death in response to DNA damage. We have previously hypothesized that the observed sensitivity of this tumor type to DNA damage is related to high basal expression of wild-type p53 protein. We have now addressed this issue by characterizing the DNA damage response of isogenic teratocarcinoma cells that differ only in their level of expression of wild-type p53 protein. We find a clear p53 dose-response relationship in these cells for rapid apoptosis following DNA damage that correlates with diminished colony formation in clonogenic survival assays. These results suggest that strategies to increase basal wild-type p53 protein expression prior to treatment with DNA-damaging drugs may improve curability in other tumor types.

Identification of partial loss of function p53 gene mutations utilizing a yeast-based functional assay.

Missense mutations within the central DNA binding region of p53 are the most prevalent mutations found in human cancer. Numerous studies indicate that 'hot-spot' p53 mutants (which comprise approximately 30% of human p53 gene mutations) are largely devoid of transcriptional activity. However, a growing body of evidence indicates that some non-hot-spot p53 mutants retain some degree of transcriptional activity in vivo, particularly against strong p53 binding sites. We have modified a previously described yeast-based p53 functional assay to readily identify such partial loss of function p53 mutants. We demonstrate the utility of this modified p53 functional assay using a diverse panel of p53 mutants.

Testicular germ cell tumors: molecular understanding and clinical implications.

It was recognized in the 1960s that testicular germ cell tumors were curable with chemotherapeutic drugs. Since that time, newer drugs including cisplatin have increased the cure rate of these tumors to over 80%, even in patients with metastatic disease. Germ cell tumors also exhibit a unique biology and genetics that distinguish them from other solid tumors and might contribute to their routine curability.

P53 tumour suppressor gene and germ cell neoplasia.

P53 tumour suppressor gene mutations occur in approximately 50% of common solid tumours such as breast, colon and lung. In contrast, p53 gene mutations occur infrequently (< 3%) in germ cell tumours, even though p53 protein is expressed at high levels in the vast majority of tumour samples. P53-regulated genes are not correspondingly over-expressed in germ cell tumour samples and cell lines, indicating that p53 functions poorly as a transcription factor in this tumour type. High levels of wild-type p53 may contribute, in part, to the chemosensitivity of germ cell tumours.

A functionally inactive p53 protein in teratocarcinoma cells is activated by either DNA damage or cellular differentiation.

Testicular teratocarcinomas never contain p53 gene mutations even though these tumors express high levels of nuclear p53 protein. We have characterized two murine teratocarcinoma cell lines and find no evidence that endogenous p53-regulated genes are correspondingly upregulated. Differentiation of these teratocarcinoma cells with retinoic acid results in a marked decrease in p53 protein levels but is accompanied by a marked increase in p53-mediated transcriptional activity. Together these results support the hypothesis that the p53 protein in undifferentiated teratocarcinoma cells is transcriptionally inactive and accounts for the lack of selection for p53 gene mutations in this tumor type. These teratocarcinoma cells undergo p53-mediated apoptosis in response to DNA damage, which may explain the routine cures of human testicular tumors with combination chemotherapy.

Secondary genomic rearrangement events in pre-B cells: VHDJH replacement by a LINE-1 sequence and directed class switching.

We describe rearrangement events which alter expression from a productive VHDJH rearrangement in an Abelson murine leukemia virus-transformed pre-B cell line. One such rearrangement results in replacement of the initially expressed variable region gene by a site-specific join between the open reading frame of a LINE-1 repetitive element and a remaining JH segment. We discuss this event in the context of the 'accessibility' model of recombinase control, and with respect to similar rearrangements involved in oncogene activation. In another subclone of the same pre-B cell line, altered heavy chain expression resulted from a mu to gamma 2b class switch recombination which occurred by a recombination-deletion mechanism but involved a complex inversion. We provide evidence that the germline gamma 2b region is specifically expressed in pre-B cell lines and early in normal development. We propose that the predisposition of pre-B cell lines to switch to gamma 2b production may reflect a normal physiological phenomenon in which the switch event is directed by an increased 'accessibility' of the germline gamma 2b locus to switch-recombination enzymatic machinery. Our findings support the hypothesis that the apparently distinct recombination systems involved in variable region gene assembly and heavy chain class switching are both directed by the accessibility of their substrate gene segments.