Wild-type p53 restores cell cycle control and inhibits gene amplification in cells with mutant p53 alleles.

Loss of cell cycle control and acquisition of chromosomal rearrangements such as gene amplification often occur during tumor progression, suggesting that they may be correlated. We show here that the wild-type p53 allele is lost when fibroblasts from patients with the Li-Fraumeni syndrome (LFS) are passaged in vitro. Normal and LFS cells containing wild-type p53 arrested in G1 when challenged with the uridine biosynthesis inhibitor PALA and did not undergo PALA-selected gene amplification. The converse occurred in cells lacking wild-type p53 expression. Expression of wild-type p53 in transformants of immortal and tumor cells containing mutant p53 alleles restored G1 control and reduced the frequency of gene amplification to undetectable levels. These studies reveal that p53 contributes to a metabolically regulated G1 check-point, and they provide a model for understanding how abnormal cell cycle progression leads to the genetic rearrangements involved in tumor progression.     

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.     

Stochastic appearance of mammary tumors in transgenic mice carrying the MMTV/c-neu oncogene

Transgenic mice carrying the activated c-neu oncogene under the control of the mouse mammary tumor virus (MMTV) long terminal repeat were produced. Epithelial hyperplasia of epididymis, seminal vesicles, and salivary glands, and dysplasia of harderian glands, were induced. Moreover, in females of our four lines, independent but multiple mammary tumors arose asynchronously, between 5 and 10 months of age, as stochastic events. Histologically, poorly differentiated adenocarcinomas, with intratumor necrosis and calcifications, arose adjacent to morphologically normal epithelium. High transgene expression was detected in all mammary tumors tested and in normal mammary glands before the appearance of the tumors. Together these results suggest that the expression of the activated c-neu oncogene was necessary but not sufficient to induce malignant transformation of the mammary epithelial cells. These tumors appear to be an adequate model for human breast cancers overexpressing c-neu.     

Abnormal adherence junctions in the heart and reduced angiogenesis in transgenic mice overexpressing mutant type XIII collagen

Type XIII collagen is a type II transmembrane protein found at sites of cell adhesion. Transgenic mouse lines were generated by microinjection of a DNA construct directing the synthesis of truncated alpha1(XIII) chains. Shortened alpha 1(XIII) chains were synthesized by fibroblasts from mutant mice, and the lack of intracellular accumulation in immunofluorescent staining of tissues suggested that the mutant molecules were expressed on the cell surface. Transgene expression led to fetal lethality in offspring from heterozygous mating with two distinct phenotypes. The early phenotype fetuses were aborted by day 10.5 of development due to a lack of fusion of the chorionic and allantoic membranes. The late phenotype fetuses were aborted by day 13.5 of development and displayed a weak heartbeat, defects of the adherence junctions in the heart with detachment of myofilaments and abnormal staining for the adherence junction component cadherin. Decreased microvessel formation was observed in certain regions of the fetus and the placenta. These results indicate that type XIII collagen has an important role in certain adhesive interactions that are necessary for normal development.     

Co-localization of mutant p53 and amyloid-like protein aggregates in breast tumors

P53 is one of the most important tumor suppressor proteins in human cancers. Mutations in the TP53 gene are common features of malignant tumors and normally correlate to a more aggressive disease. In breast cancer, these gene alterations are present in approximately 20% of cases and are characteristically of missense type. In the present work we describe TP53 mutations in breast cancer biopsies and investigate whether wild and mutant p53 participate in protein aggregates formation in these breast cancer cases. We analyzed 88 biopsies from patients residing in the metropolitan area of Rio de Janeiro, and performed TP53 mutation screening using direct sequencing of exons 5-10. Seventeen mutations were detected, 12 of them were of missense type, 2 nonsenses, 2 deletions and 1 insertion. The presence of TP53 mutation was highly statistically associated to tumor aggressiveness of IDC cases, indicated here by Elston Grade III (p<0.0001). Paraffin embedded breast cancer tissues were analyzed for the presence of p53 aggregates through immunofluorescence co-localization assay, using anti-aggregate primary antibody A11, and anti-p53. Our results show that mutant p53 co-localizes with amyloid-like protein aggregates, depending on mutation type, suggesting that mutant p53 may form aggregates in breast cancer cells, in vivo.     

Integration of Friend murine leukemia virus into both alleles of the p53 oncogene in an erythroleukemic cell line.

The Friend virus-transformed erythroleukemic cell line DP16-9B4 has undergone a complex rearrangement of the p53 oncogene and lacks any detectable expression of the p53 protein. We report here characterization of both p53 alleles in this cell line and identify independent integrations of Friend murine leukemia virus sequences into the coding region of both alleles.     

Retention of wild-type p53 in tumors from p53 heterozygous mice: reduction of p53 dosage can promote cancer formation.

Tumor suppressor genes are generally viewed as being recessive at the cellular level, so that mutation or loss of both tumor suppressor alleles is a prerequisite for tumor formation. The tumor suppressor gene, p53, is mutated in approximately 50% of human sporadic cancers and in an inherited cancer predisposition (Li-Fraumeni syndrome). We have analyzed the status of the wild-type p53 allele in tumors taken from p53-deficient heterozygous (p53+/-) mice. These mice inherit a single null p53 allele and develop tumors much earlier than those mice with two functional copies of wild-type p53. We present evidence that a high proportion of the tumors from the p53+/- mice retain an intact, functional, wild-type p53 allele. Unlike p53+/- tumors which lose their wild-type allele, the tumors which retain an intact p53 allele express p53 protein that induces apoptosis following gamma-irradiation, activates p21(WAF1/CIP1) and Mdm2 expression, represses PCNA expression (a negatively regulated target of wild-type p53), shows high levels of binding to oligonucleotides containing a wild-type p53 response element and prevents chromosomal instability as measured by comparative genomic hybridization. These results indicate that loss of both p53 alleles is not a prerequisite for tumor formation and that mere reduction in p53 levels may be sufficient to promote tumorigenesis.     

A role of cytoplasmic p53 in the regulation of metabolism shown by bat-mimicking p53 NLS mutant mice

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Tumor suppressor p53: analysis of wild-type and mutant p53 complexes.

It has been suggested that the dominant effect of mutant p53 on tumor progression may reflect the mutant protein binding to wild-type p53, with inactivation of suppressor function. To date, evidence for wild-type/mutant p53 complexes involves p53 from different species. To investigate wild-type/mutant p53 complexes in relation to natural tumor progression, we sought to identify intraspecific complexes, using murine p53. The mutant phenotype p53-246(0) was used because this phenotype is immunologically distinct from wild-type p53-246+ and thus permits immunological analysis for wild-type/mutant p53 complexes. The p53 proteins were derived from genetically defined p53 cDNAs expressed in vitro and also from phenotypic variants of p53 expressed in vivo. We found that the mutant p53 phenotype was able to form a complex with the wild type when the two p53 variants were cotranslated. When mixed in their native states (after translation), the wild-type and mutant p53 proteins did not exhibit any binding affinity for each other in vitro. Under identical conditions, complexes of wild-type human and murine p53 proteins were formed. For murine p53, both the wild-type and mutant p53 proteins formed high-molecular-weight complexes when translated in vitro. This oligomerization appeared to involve the carboxyl terminus, since truncated p53 (amino acids 1 to 343) did not form complexes. We suggest that the ability of the mutant p53 phenotype to complex with wild type during cotranslation may contribute to the transforming function of activated mutants of p53 in vivo.     

Increased epidermal tumors and increased skin wound healing in transgenic mice overexpressing the catalytic subunit of telomerase, mTERT, in basal keratinocytes

Telomerase transgenics are an important tool to assess the role of telomerase in cancer, as well as to evaluate the potential use of telomerase for gene therapy of age-associated diseases. Here, we have targeted the expression of the catalytic component of mouse telomerase, mTERT, to basal keratinocytes using the bovine keratin 5 promoter. These telomerase-transgenic mice are viable and show histologically normal stratified epithelia with high levels of telomerase activity and normal telomere length. Interestingly, the epidermis of these mice is highly responsive to the mitogenic effects of phorbol esters, and it is more susceptible than that of wild-type littermates to the development skin tumors upon chemical carcinogenesis. The epidermis of telomerase-transgenic mice also shows an increased wound-healing rate compared with wild-type littermates. These results suggest that, contrary to the general assumption, telomerase actively promotes proliferation in cells that have sufficiently long telomeres and unravel potential risks of gene therapy for age-associated diseases based on telomerase upregulation.     

Early postnatal cardiac changes and premature death in transgenic mice overexpressing a mutant form of serum response factor

Serum response factor (SRF) is a key regulator of a number of extracellular signal-regulated genes important for cell growth and differentiation. A form of the SRF gene with a double mutation (dmSRF) was generated. This mutation reduced the binding activity of SRF protein to the serum response element and reduced the capability of SRF to activate the atrial natriuretic factor promoter that contains the serum response element. Cardiac-specific overexpression of dmSRF attenuated the total SRF binding activity and resulted in remarkable morphologic changes in the heart of the transgenic mice. These mice had dilated atrial and ventricular chambers, and their ventricular wall thicknesses were only 1/2 to 1/3 the thickness of that of nontransgenic mice. Also these mice had smaller cardiac myocytes and had less myofibrils in their myocytes relative to nontransgenic mice. Altered gene expression and slight interstitial fibrosis were observed in the myocardium of the transgenic mice. All the transgenic mice died within the first 12 days after birth, because of the early onset of severe, dilated cardiomyopathy. These results indicate that dmSRF overexpression in the heart apparently alters cardiac gene expression and blocks normal postnatal cardiac growth and development.     

Two distinct Notch1 mutant alleles are involved in the induction of T-cell leukemia in c-myc transgenic mice

We have previously characterized a large panel of provirus insertion Notch1 mutant alleles and their products arising in thymomas of MMTV(D)/myc transgenic mice. Here, we show that these Notch1 mutations represent two clearly distinct classes. In the first class (type I), proviral integrations were clustered just upstream of sequences encoding the transmembrane domain. Type I Notch1 alleles produced two types of mutant Notch1 RNA, one of which encoded the entire Notch1 cytoplasmic domain [N(IC)] and the other of which encoded a soluble ectodomain [N(EC)(Mut)] which, in contrast to the processed wild-type ectodomain [N(EC)(WT)], did not reside at the cell surface and became secreted in a temperature-dependent manner. A second, novel class of mutant Notch1 allele (type II) encoded a Notch1 receptor with the C-terminal PEST motif deleted (DeltaCT). The type II Notch1(DeltaCT) protein was expressed as a normally processed receptor [N(EC)(WT) and N(IC)(DeltaCT)] at the cell surface, and its ectodomain was found to be shed into the extracellular medium in a temperature- and calcium-dependent manner. These data suggest that both type I and type II mutations generate two structurally distinct Notch1 N(EC) and N(IC) proteins that may participate in tumor formation, in collaboration with the c-myc oncogene, through distinct mechanisms. Constitutive type I N(IC) and type II N(IC)(DeltaCT) expression may enhance Notch1 intracellular signaling, while secreted or shed type I N(EC)(Mut) and type II N(EC) proteins may differentially interact in an autocrine or paracrine fashion with ligands of Notch1 and affect their signaling.     

Cyclic hair-loss and regrowth in transgenic mice overexpressing an intermediate filament gene.

We have produced transgenic mice containing up to 250 copies of a sheep wool intermediate filament keratin gene to study the effect of its expression on hair structure and development. Several transgenic lines expressed the gene and in the one containing 250 transgenes, a pattern of hair-loss and regrowth was stably established. Successive waves of hair growth follow periods of denuding like the natural progression of hairs in the mouse hair cycle. By in situ hybridization we have shown that the sheep transgenes are expressed at the correct stage in mouse hair development and at a high level. The transgenic hairs contain not only an elevated level of intermediate filament keratin protein but also a decreased level of the filament-associated proteins. This imbalance disrupts the normal ordered array of these proteins in the cells of the hair cortex and leads to weakened fibres which are prematurely lost.