Single-nucleotide polymorphisms in the p53 pathway.

A cell culture assay has been developed that detects and validates single-nucleotide polymorphisms (SNPs) in genes that populate the p53 pathway. One hundred thirteen EBV-transformed human B-lymphocyte cell lines obtained from a diverse population were employed to measure the apoptotic response to gamma radiation. Each cell line undergoes a reproducible, characteristic frequency of apoptosis, and the response of the population forms a normal distribution around a median of 35.5% apoptosis with a range from 12% to 58% apoptosis. Polymorphisms in the AKT1 and Perp genes significantly affect the frequency of apoptosis. The assay can detect both racial and sexual dimorphisms in these genes and has the ability to demonstrate epistatic relationships within the p53 pathway. The cell lines used in this assay provide biological materials to explore the molecular basis of the polymorphisms.

A male germ cell tumor-susceptibility-determining locus, pgct1, identified on murine chromosome 13.

Inbred 129 strain mice are predisposed to developing male germ cell tumors (GCTs) of the testes. The inherent genetic defects that underlie male GCT susceptibility in the 129 mouse strain are unknown. GCT incidence is increased in 129 strain males that lack functional p53 protein, and we have used this finding to facilitate the generation of panels of GCT-bearing intercross and backcross mice for genetic mapping analysis. A 129 strain locus, designated pgct1, that segregates with the male GCT phenotype has been identified on chromosome 13 near D13Mit188. This region of murine chromosome 13 may be syntenic to a portion of human chromosome 5q that is implicated in male GCT susceptibility in humans.

Genetic mapping of the embryonal carcinoma transplantation resistance locus Gt(B6) to mouse Chromosome 8.

Cytotoxic T lymphocytes play a predominant role in allograft rejection. They mediate this process through recognition of foreign major histocompatibility complex (MHC) class I surface molecules encoded at the H2 locus. Embryonal carcinoma cells, the undifferentiated, neoplastic derivatives of primordial germ cells, typically lack detectable MHC class I gene expression. Despite this, embryonal carcinoma cells are subject to allograft rejection in several different mouse strains. In many instances, the H2 locus appears to be genetically linked to resistance. However, rejection of allografts of the F9 embryonal carcinoma cell line, a nullipotent cell line derived from the 129 mouse strain, does not appear to be H2-linked. Resistance to F9 tumor formation in the C57BL/6 mouse strain has been attributed to a single, unidentified locus termed Gt(B6). To genetically map the Gt(B6) locus, a total of 463 (C57BL/6x129)F2 mice were challenged with F9 cells, and 78 tumor-resistant mice were identified. Markers encompassing two candidate regions, the H2 locus on Chromosome (Chr) 17 and a second candidate locus on Chr 2, showed no indication of linkage to the resistance phenotype. Instead, results of a genome wide scan implicated mouse Chr 8, and evidence is presented demonstrating that the Gt(B6) locus maps to a region of less than 10 cM on the medial portion of Chr 8.

Cytotoxic T-lymphocyte epitope immunodominance in the control of choroid plexus tumors in simian virus 40 large T antigen transgenic mice.

The simian virus 40 (SV40) large tumor antigen (Tag) is a virus-encoded oncoprotein which is the target of a strong cytotoxic T-lymphocyte (CTL) response. Three immunodominant H-2(b)-restricted epitopes, designated epitopes I, II/III, and IV, have been defined. We investigated whether induction of CTLs directed against these Tag epitopes might control Tag-induced tumors in SV11(+) (H-2(b)) mice. SV11(+) mice develop spontaneous tumors of the choroid plexus due to expression of SV40 Tag as a transgene. We demonstrate that SV11(+) mice are functionally tolerant to the immunodominant Tag CTL epitopes. CTLs specific for the H-2Kb-restricted Tag epitope IV were induced in SV11(+) mice following adoptive transfer with unprimed C57BL/6 spleen cells and immunization with recombinant vaccinia viruses expressing either full-length Tag or the H-2Kb-restricted epitope IV as a minigene. In addition, irradiation of SV11(+) mice prior to adoptive transfer with unprimed C57BL/6 spleen cells led to the priming of epitope IV-specific CTLs by the endogenous Tag. Induction of epitope IV-specific CTLs in SV11(+) mice by either approach correlated with increased life span and control of the choroid plexus tumor progression, indicating that CTLs specific for the immunodominant Tag epitope IV control the progressive growth of spontaneous tumors induced by this DNA virus oncogene in transgenic mice.

Two critical hydrophobic amino acids in the N-terminal domain of the p53 protein are required for the gain of function phenotypes of human p53 mutants.

Some mutant forms of the p53 protein have been shown to gain new functions that are not shared by the wild-type p53 protein; (1) mutant p53 proteins can transcriptionally transactivate the multi-drug resistance gene-1 (MDR-1) and (2) when expressed in non-tumorigenic cells with no endogenous p53 protein, mutant p53 proteins can enhance the tumorigenic potential of these cells (Dittmer et al., 1993). It has recently been shown (Lin et al., 1994b) that the transcriptional activator domain of the p53 protein contains two amino acids, leu-22 and trp-23, which are required by the wild-type p53 protein for transcriptional activity. To determine whether these same amino acid residues are utilized by mutant p53 proteins for their gain of function phenotype, the triple mutant p53 protein (at residues 22 and 23 in the transactivation domain and residue 281 in the DNA binding domain--a gain of function mutant) was made. While the p53-281 mutant transcriptionally activates the MDR-1 gene and enhances the tumorigenic potential of cells it is expressed in, the 22, 23, 281 triple mutant failed to carry out either of these functions.

Gain of function mutations in p53.

We report that the expression of murine or human mutant p53 proteins in cells with no endogenous p53 proteins confers new or additional phenotypes upon these cells. Mutant p53 proteins expressed in cell lines lacking p53 resulted in either enhanced tumorigenic potential in nude mice ((10)3 cells) or enhanced plating efficiency in agar cell culture (human SAOS-2 cells). Also, mutant human p53 alleles, unlike the wild-type p53 protein, could also enhance the expression of a test gene regulated by the multi-drug resistance enhancer-promoter element. These data demonstrate a gain of function associated with p53 mutations in addition to the loss of function shown previously to be associated with mutations in this tumour suppressor gene.

Antibody response to human papovavirus JC (JCV) and simian virus 40 (SV40) T antigens in SV40 T antigen-transgenic mice.

Human papovavirus JC (JCV) and simian virus 40 (SV40) genomes share approximately 69% homology; and there is antigenic cross-reactivity between JCV and SV40 tumor or T antigens. In order to determine whether a selective immune response to JCV T antigen could be demonstrated, transgenic mice (SV11+) that express SV40 T antigen in the choroid plexus and are partially tolerant to antigenic determinants on SV40 T antigen were immunized with SV40 or JCV T antigens and their antibody responses were analyzed. The results show that SV11+ mice responded as well as their nontransgenic litter mates to JCV T antigen. Monoclonal antibodies were derived from hybridomas generated from immunized mice which reacted specifically with epitopes in the amino and carboxy terminal halves on JCV T antigen. These studies show that transgenic mice expressing SV40 T antigen are capable of responding to determinants not shared between JCV and SV40 T antigen.

p53 mutations are not selected for in simian virus 40 T-antigen-induced tumors from transgenic mice.

Many diverse tumors contain cells that select for mutations at the p53 gene locus. This appears to be the case because the p53 gene product can act as a negative regulator of cell division or a tumor suppressor. These mutations then eliminate this activity of the p53 gene product. The simian virus 40 (SV40) large T antigen binds to p53 and acts as an oncogene to promote cellular transformation and initiate tumors. If the binding of T antigen to the p53 protein inactivated its tumor suppressor activity, there would be no selection pressure for p53 mutants to appear in tumors. To test this idea, transgenic mice that carried and expressed the SV40 large T-antigen gene were created. Expression of the T antigen was directed to the liver, using the albumin promoter, and the choroid plexus, using the SV40 enhancer-promoter. A large number of papillomas (indicated in parentheses) of the choroid plexus (14), hepatocellular carcinomas (5), liver adenomas (10), and tumors of clear-cell foci (5) were examined for mutant and wild-type p53 genes and gene products. In all cases, the tumor extracts contained readily detectable T-antigen-p53 protein complexes. A monoclonal antibody specifically recognizing the wild-type p53 protein (PAb246) reacted with p53 in every tumor extract. A monoclonal antibody specifically recognizing mutant forms of the p53 protein (PAb240) failed to detect p53 antigens in these extracts. Finally, p53 partial cDNAs were sequenced across the regions of common mutations in this gene, and in every case only the wild-type sequence was detected. These results strongly support the hypothesis that T antigen inactivates the wild-type p53 tumor-suppressing activity and there is no need to select for mutations at the p53 locus.

Impact of the genetic background of transgenic mice upon the formation and timing of choroid plexus papillomas.

Transgenic mice harboring the SV40 large T antigen gene in a C57B1/6J genetic background (SV11) first express this gene at 1-2 weeks of age, develop papillomas of the choroid plexus by 80-90 days, and die within 125 days after birth. Transgenic mice harboring the same transgene in a (SV11-C57Bl/6J x NZW/lacJ) F1 genetic background express considerably lower levels of the transgene mRNA at comparable times after birth. As a consequence, tumor development and death are delayed. The NZW mice appear to contribute a dominant negative regulator for the expression of the SV40 large T antigen transgene, which in turn has a dramatic effect upon the time of appearance of tumors and the death of these transgenic animals.

Teratocarcinoma transplantation rejection loci: genetic localization of the Gt-1 locus on chromosome 17 and the expression of alternate alleles.

A series of congenic mice on the BALB/c genetic background have been employed to localize a teratocarcinoma transplantation rejection locus, Gt-1, to the K side of the H-2 locus on chromosome 17. Previous studies have placed the Gt-1sv allele about 8 centimorgans away from the H-2b or H-2bv1 locus. Teratocarcinomas derived from 129/sv mice, Gt-1sv (H-2Kbv1/H-2Dbv1), are rejected by BALB/c(H-2Kd/H-2Dd) and BALB.G mice (H-2Kd/H2D-b, but form tumors in BALB.B (H-2Kb/H2Db) and BALB/5R5 mice (H-2Kb/H2Dd). In the reciprocal tumor-rejection test, a BALB/c teratocarcinoma was rejected by immunized BALB.B mice, but formed tumors in the immunized isogenic BALB/c mouse. These studies demonstrate the reciprocal expression of two Gt-1 alleles, one Gt-1c, in BALB/c mice, and the other, Gt-1sv, in the congenic BALB.B mice, Shedlovsky and co-workers have placed the GT-1 locus in a similar location on the K side of the H-2 locus on chromosome 17.

In vitro differentiation of teratomas and the distribution of creatine phosphokinase and plasminogen activator in teratocarcinoma-derived cells.

Mouse teratocarcinoma cells from embryoid bodies were cultured in vitro to permit their differentiation into a number of cell types. Two enzyme activities, creatine phosphokinase (CPK) and the protease plasminogen activator, were studied to follow the developmental sequence of events in these embryoid body-derived cell cultures. CPK activity increased with time in culture, indicating the appearance of new cell types with brain- or muscle-specific enzyme activities. Plasminogen activator was detectable in extracts of embryoid bodies. This protease activity first increased and then decreased to a low level as the embryoid bodies in culture developed into differentiated cell types. These cell cultures also showed a decreased potential for tumor formation in syngeneic mice as a function of time in culture. This decrease in tumorigenic potential was correlated with the appearance of differentiated cells in vitro. Simian virus 40 (SV40) was used to infect and transform cells derived from embryoid bodies in culture. This was done to permit the establishment of cloned teratocarcinoma-derived cell lines. Twenty-nine distinct cloned permanent cell lines (called SVTER) containing the SV40-specific tumor antigen were obtained. None of these cell lines was capable of producing tumors in syngeneic mice. An analysis of the levels of creatine phosphokinase and plasminogen activator in these SVTER cell lines indicated that : (a) some cell lines had high CPK activity and little or no plasminogen activator activity, (b) some cell lines contained high levels of plasminogen activator activity with little or no CPK activity, and (c) some cell lines contained neither of these enzyme activities. No example of a cell line with high levels of both enzyme activities was observed, indicating that these two enzymes may participate in mutually exclusive developmental pathways. The SVTER cell lines may therefore be useful in reconstructing these developmental pathways in vitro.

Deoxyribonucleic acid replication in simian virus 40-infected cells. II. Detection and characterization of simian virus 40 pseudovirions.

Purified simian virus 40 (SV40) virions, grown in primary African green monkey kidney cells labeled prior to infection with (3)H-thymidine, contain a variable quantity of (3)H-labeled deoxyribonucleic acid (DNA). This DNA is resistant to deoxyribonuclease, sediments at 250S, and is enclosed in a particle that can be precipitated with SV40-specific antiserum. DNA-DNA hybridization experiments demonstrate that this (3)H-labeled component in purified SV40 virions is cellular DNA. When this (3)H-labeled DNA is released from purified virus with sodium dodecyl sulfate, it has an average sedimentation constant of 14S. Sedimentation through neutral and alkaline sucrose gradients shows that this 14S DNA is composed of a collection of different sizes of DNA molecules that sediment between 11 and 15S. As a result of this size heterogeneity, SV40 virions containing cellular DNA (pseudovirions) have a variable DNA to capsid protein ratio and exhibit a spectrum of buoyant densities in a CsCl equilibrium gradient. Pseudovirions are enriched, relative to true virions, on the lighter density side of infectious SV40 virus banded to equilibrium in a CsCl gradient. Little or no cellular DNA was found in purified SV40 virus preparations grown in BSC-1 or CV-1 cells.