p53-deficient mice are protected against adrenalectomy-induced apoptosis

The p53 tumor suppressor gene, an important regulator of the cell cycle, has been implicated in apoptotic cell death in vitro, and more recently in neuronal degeneration in vivo. The present study investigated the importance of p53 expression in the apoptotic death of hippocampal granule cells following adrenalectomy. Mice, either homozygous or heterozygous for the p53 null allele and wild-type controls were sacrificed 16 days after adrenalectomy. Hippocampal morphology was assessed in paraffin sections stained with hematoxylin and eosin. Cells exhibiting features characteristic of apoptosis were evident in hippocampi from wild-type mice. A significant decrease in the number of apoptotic cells was observed in both homozygous and heterozygous mice. These findings demonstrate that absence or attenuation of p53 expression protects granule cells from adrenalectomy-induced apoptosis and, combined with the results of other studies, suggest that p53 is required for certain types of neuronal degeneration.     

Establishment of uterine cell lines from p53-deficient mice

OBJECTIVE: To evaluate the relationship of E-cadherin (E-CD) expression to cellular DNA content and biological behavior of gastric cancer. METHODS: E-CD expression and cellular DNA content were quantitatively measured by flow cytometry and immunofluorescence methods in 80 cases of formalin-fixed, paraffin-embedded gastric cancer. Systematic pathological examinations and follow up were performed for all cases. RESULTS: E-CD expression was significantly reduced in all cases of gastric cancer, fluorescence Index (FI) of E-CD expression was 0.67 +/- 0.11 in gastric cancer, 1.0 +/- 0.07 in normal gastric mucosa (P < 0.001). The reduction of E-CD expression was also found in 2 cases of early gastric cancer. Tumors with a decrease in E-CD expression occurred significantly more frequently in undifferentiated, diffuse growth pattern Borrmann 4 type, positive lymph node (LN) metastasis and infiltrated serosa type gastric cancer, of which survival time was within 5 years (P < 0.001). E-CD expression was also reduced in gastric cancer with the number of LN metastasis above 5, metastasis to more than group 2. E-CD expression was lower in uneuploid cancer than that in diploid cancer (P < 0.01). The value of DI and PI with negative E-CD expression was significantly higher than that of positive E-CD expression (P < 0.01). CONCLUSION: Down-regulation of E-CD expression correlates with bad biological behavior and poor prognosis of gastric cancer. The reduction of E-CD expression may take place during early time of gastric cancer. Quantitative analysis of E-CD expression may have some value in evaluating the intensity of LN metastasis of gastric cancer.     

Thymic stromal-cell abnormalities and dysregulated T-cell development in IL-2-deficient mice

Vineland Adaptive Behavior Scales Special Population norms are presented for four groups of individuals with autism: (a) mute children under 10 years of age; (b) children with at least some verbal skills under 10 years of age; (c) mute individuals who are 10 years of age or older; and (d) individuals with at least some verbal skills who are 10 years of age or older. The sample included 684 autistic individuals ascertained from cases referred for the DSM-IV autism/PDD field trial collaborative study and five university sites with expertise in autism. Young children had higher standard scores than older individuals across all Vineland domains. In the Communication domain, younger verbal children were least impaired, older mute individuals most impaired, and younger mute and older verbal individuals in the midrange. Verbal individuals achieved higher scores in Daily Living Skills than mute individuals. The expected profile of a relative weakness in Socialization and relative strength in Daily Living Skills was obtained with age-equivalent but not standard scores. Results high-light the importance of employing Vineland special population norms as well as national norms when evaluating individuals with autism.     

Dental innervation and CGRP in adult p75-deficient mice

Adult dental tissues have unusual neurotrophin biology. Pulpal fibroblasts express nerve growth factor (NGF) and the low-affinity p75 neurotrophin receptor, their sensory nerve fibers express p75 and trk A, and pulpal sympathetic fibers lack p75. Following tooth injury, there is increased pulpal NGF, sprouting of sensory nerve endings, and increased immunoreactivity for the sensory neuropeptide calcitonin gene-related peptide (CGRP). In the present study, we have analyzed tooth structure and innervation of pulp and periodontal ligament in young (6-8 weeks, 3 months) and older (5-12 months) adult mice carrying a null mutation in the p75 gene and compared the results with those of age-matched wild-type controls. Our hypotheses were that tooth structure would be abnormal and that pulpal innervation would be greatly reduced because it consists primarily of nociceptive fibers that have been found to be severely depleted in skin of p75(-/-) mice. Tissues were fixed, X-rayed for gross dental morphology, decalcified, and analyzed for immunoreactivity for CGRP and for a general nerve marker, protein gene product 9.5. Radiographs showed worn-down molar crowns in p75-deficient mice. Light microscopy confirmed the accelerated molar wear and showed intense CGRP immunoreactivity in pulp nerve endings of mutant mice, compared with a gradual decrease in CGRP intensity in controls during normal aging. The CGRP intensity in 5-12-month-old pairs of mice was threefold greater in the mutants (P < 0.03), and in younger mice the mutant always had more CGRP than its matched control. The innervation of molar ligament in all p75-deficient mice was similar to that of controls except there was nerve sprouting near bone loss in mutants. The incisors of mutant mice did not have unusual wear and their pulpal CGRP immunoreactivity remained normal, but their periodontal ligament had fewer thin branched nerve endings at all ages. Thus, most innervation of teeth and their supporting tissues developed normally, and the only neural changes in p75(-/-) mutant mice were the reduction of incisor ligament sensory receptors and increased molar CGRP. Sensory nerves in teeth gradually lose neuropeptide intensity during aging, but that did not happen in the mutant mice, suggesting that the accelerated molar wear stimulated persistent high levels of CGRP.     

Cyclin-dependent kinase 5-deficient mice demonstrate novel developmental arrest in cerebral cortex

The cerebral cortex of mice with a targeted disruption in the gene for cyclin-dependent kinase 5 (cdk5) is abnormal in its structure. Bromodeoxyuridine labeling reveals that the normal inside-out neurogenic gradient is inverted in the mutants; earlier born neurons are most often found superficial to those born later. Despite this, the early preplate layer separates correctly and neurons with a normal, pyramidal morphology can be found between true marginal zone and subplate. Consistent with their identity as layer VI corticothalamic neurons, they can be labeled by DiI injections into thalamus. The DiI injections also reveal that the trajectories of the cdk5(-/-) thalamocortical axons are oblique and cut across the entire cortical plate, instead of being oriented tangentially in the subcortical white matter. We propose a model in which the cdk5(-/-) defect blocks cortical development at a heretofore undescribed intermediate stage, after the splitting of the preplate, but before the migration of the full complement of cortical neurons.     

An Alzheimer's disease-linked PS1 variant rescues the developmental abnormalities of PS1-deficient embryos

Mutations in presenilin 1 (PS1) cosegregate with approximately 25% of early onset familial Alzheimer's disease (FAD) pedigrees. A variety of in vitro and in vivo paradigms have established that one mechanism by which PS1 variants cause AD is by elevating the production of highly amyloidogenic Abeta1-42/43 peptides. PS1 is homologous to sel-12, a C. elegans protein that facilitates signaling mediated by the Notch/lin-12 family of receptors. Wild-type human PS1 complements an egg-laying defect in C. elegans lacking sel-12, while FAD-linked PS1 variants exhibit reduced rescue activity. These data suggested that mutant PS1 may cause disease as a result of reduction in PS1 function. To test the function of FAD-linked PS1 in mammals, we examined the ability of the A246E PS1 variant to complement the embryonic lethality and axial skeletal defects in mice lacking PS1. Finally, to examine the influence of reduced PS1 levels on Abeta production, we quantified Abeta1-42/43 peptide levels in PS1 heterozygous null mice (PS1[+/-] mice). We now report that both human wild-type and A246E PS1 efficiently rescue the phenotypes observed in PS1(-/-) embryos, findings consistent with the view that FAD-linked PS1 mutants retain sufficient normal function during mammalian embryonic development. Moreover, the levels of Abeta1-42/43 and Abeta1-40 peptides between PS1(+/-) and control mice are indistinguishable. Collectively, these data lead us to conclude that mutant PS1 causes AD not by loss of normal PS1 function but by influencing amyloid precursor protein (APP) processing in a manner that elevates Abeta1-42/43 production.     

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 fraction unresponsive to growth inhibition by TGF-beta among the high-proliferative potential progenitor cells in bone marrow of p53-deficient mice

The interaction between the respiratory pathogen Bordetella bronchiseptica and epithelial cells was studied. After 2-3 h, B. bronchiseptica strains exerted a strong cytotoxic effect on HeLa cells which was evident by rounding of the cells and detachment from the substrate and which ultimately resulted in total disintegration of the host cell. Production of this cytotoxic activity appeared to be regulated by the BvgAS sensory transduction system, which coordinately regulates many B. bronchiseptica virulence factors, since bacteria cultured in the presence of sulfate anions, inhibitors of the BvgAS response, did not exhibit this effect. Furthermore, spontaneous phase variants of B. bronchiseptica strains adhered to HeLa cells but were not cytotoxic. The cytotoxic component is presumably not secreted because the bacterial culture supernatant was not cytotoxic for HeLa cells. Besides HeLa cells other human epithelial cell lines such as Chang cells, larynx HEp-2 cells and lung NCI-H292 cells were sensitive to the cytotoxic activity of B. bronchiseptica. These results suggest the presence of a novel BvgAS-regulated virulence factor in B. bronchiseptica.     

Comparison of p53 gene abnormalities in bilateral and unilateral breast cancer

BACKGROUND: The results of recent studies have suggested that p53 gene abnormalities are associated with carcinogenesis in several neoplasms. It is believed that bilateral breast carcinomas develop as a result of a different carcinogenetic mechanism and genetic environment from those of unilateral lesions. METHODS: p53 Gene abnormalities in bilateral primary breast cancer were detected by polymerase chain reaction-single strand comformation polymorphism (PCR-SSCP) analysis. A total of 76 paraffin embedded tissue specimens from 38 patients with bilateral primary breast cancer were examined, and 62 patients with unilateral breast cancer were analyzed as control subjects. The bilateral tumors were defined as primary, based on clinical parameters and the presence of an intraductal component. There were 13 patients with synchronous bilateral breast cancer and 25 with metachronous bilateral breast cancer. RESULTS: p53 Gene abnormalities were detected in 50% of the bilateral and 25.8% of the unilateral cases, and the difference was significant (P < 0.01, chi-square test). Abnormalities were detected in 56% of the metachronous cases, representing a much higher incidence than that of the unilateral cases (P < 0.001, chi-square test). The incidence of p53 gene abnormalities in the first and second tumors of the metachronous cases was 44% and 68%, respectively. The percentage of patients with a p53 gene abnormality and positive family history was higher for those with bilateral than with unilateral breast cancer (P < 0.01, chi-square test). CONCLUSION: These findings indicate that the genetic changes and mechanism of carcinogenesis in bilateral and unilateral breast cancer are different.     

Blastic transformation of p53-deficient bone marrow cells by p210bcr/abl tyrosine kinase

Blastic transformation of chronic myelogenous leukemia (CML) is characterized by the presence of nonrandom, secondary genetic abnormalities in the majority of Philadelphia1 clones, and loss of p53 tumor suppressor gene function is a consistent finding in 25-30% of CML blast crisis patients. To test whether the functional loss of p53 plays a direct role in the transition of chronic phase to blast crisis, bone marrow cells from p53+/+ or p53-/- mice were infected with a retrovirus carrying either the wild-type BCR/ABL or the inactive kinase-deficient mutant, and were assessed for colony-forming ability. Infection of p53-/- marrow cells with wild-type BCR/ABL, but not with the kinase-deficient mutant, enhanced formation of hematopoietic colonies and induced growth factor independence at high frequency, as compared with p53+/+ marrow cells. These effects were suppressed when p53-/- marrow cells were coinfected with BCR/ ABL and wild-type p53. p53-deficient BCR/ABL-infected marrow cells had a proliferative advantage, as reflected by an increase in the fraction of S+G2 phase cells and a decrease in the number of apoptotic cells. Immunophenotyping and morphological analysis revealed that BCR/ABL-positive p53-/- cells were much less differentiated than their BCR/ABL-positive p53+/+ counterparts. Injection of immunodeficient mice with BCR/ABL-positive p53-/- cells produced a transplantable, highly aggressive, poorly differentiated acute myelogenous leukemia. In marked contrast, the disease process in mice injected with BCR/ABL-positive p53+/+ marrow cells was characterized by cell infiltrates with a more differentiated phenotype and was significantly retarded, as indicated by a much longer survival of leukemic mice. Together, these findings directly demonstrate that loss of p53 function plays an important role in blast transformation in CML.     

The p53-deficient mouse: a model for basic and applied cancer studies

Inactivation of the p53 gene in the germline of mice by gene targeting has provided researchers with a model similar in many respects to the analogous human inherited cancer predisposition Li-Fraumeni syndrome. The viability of p53 null mice has allowed unexpected opportunities to study the role of p53 in many different in-vivo and in-vitro contexts. Null (p53-/-) mice have an average time to tumor development of 4.5 months, while half of the heterozygous (p53+/-) mice develop tumors by 18 months. The p53-deficient mice have been particularly valuable in examining the effects of p53 loss on tumor progression. In addition, the mice hold significant promise as tools to assess carcinogens, teratogens, chemopreventative agents, and cancer therapeutic regimens.     

Analysing human developmental abnormalities

About one in forty babies is born with a recognisable congenital anomaly at birth. Rapid progress is being made in recognising the genetic contribution to these defects. From over 2000 likely single gene malformation syndromes in humans the gene has been isolated or mapped in about 10%. Despite the availability of animal models, the study of malformations in humans continues to reveal novel genes and unpredicted functions for known genes. The importance of the study of clinical malformations to the understanding of embryological development in humans and other organisms is discussed and reviewed.     

p53 abnormalities in CLL are associated with excess of prolymphocytes and poor prognosis

To determine the role of the p53 gene in chronic lymphocytic leukaemia (CLL) and its possible involvement in the pathogenesis of a progressive form of CLL characterized by > 10%, prolymphocytes (CLL/PL), we selected 32 cases, 17 with typical morphology and 15 CLL/PL. The extent of inactivation of p53 was examined by assessing loss of heterozygosity (LOH) at 17p13.3, by sequencing the highly conserved region (exons 5-9) of the p53 gene and by analysing p53 protein expression. LOH was detected in 8/28 (29%) cases, p53 mutations in 5/32 (16%) cases and p53 expression in 5/27 (19%) cases. Overall 11 cases (30%) had p53 abnormalities of which eight cases had CLL/PL. There was a significant association between CLL/PL and p53 abnormalities (P=0.05); 75% of cases with LOH, 80% of p53 mutations and 80% of cases positive for p53 protein had CLL/PL. Thus, p53 inactivation is the first gene abnormality identified so far to be involved in the development of CLL/PL. All the cases with typical CLL and p53 abnormalities had only one allele affected whereas 4/6 CLL/PL had both alleles inactivated. This difference in the extent of p53 inactivation suggests that accumulation of p53 abnormalities may be associated with progression of CLL to CLL/PL. CLL cases with p53 abnormalities were characterized by a higher incidence of stage C (P<0.025), a higher proliferative rate (P=0.05), short survival (P<0.005) and resistance to first-line therapy (P<0.02) but not to nucleoside analogues. Analysis of the correlation between p53 status and incidence of trisomy 12 by fluorescence in situ hybridization (FISH) showed that trisomy 12 was more frequent in cases without p53 abnormalities, suggesting that trisomy 12 and p53 may represent different pathways of transformation in CLL.     

Analysis of centrosome abnormalities and angiogenesis in epidermal-targeted p53172H mutant and p53-knockout mice after chemical carcinogenesis: evidence for a gain of function

Genetic defects of the human androgen receptor (AR) can cause a wide spectrum of androgen insensitivity syndromes (AIS) in XY individuals ranging from phenotypic females, to defective spermatogenesis in otherwise normal males. We screened the non-polymorphic regions of exon 1, transactivation domain (TAD), of the AR gene in 153 subjects with varying degrees of defective spermatogenesis of unknown aetiology, and compared them to 100 healthy fertile controls. Three different single-strand conformation polymorphisms were detected and sequencing of the mutant fragments revealed three G-->A transitions in codons 210, 211 and 214. The first two mutations were polymorphisms and the transition in codon 211 was related to ethnic origin occurring in 10-15% of Indian or Middle-Eastern subjects, but not in the majority of Chinese. The third mutation resulted in a non-conservative glycine to arginine substitution at codon 214 (G214R) and was associated with approximately 20% lower transactivation capacity compared to the wild-type (WT). This study, the first screening of the AR TAD for subtle mutations, in a large group of males with defective spermatogenesis, has uncovered novel polymorphisms which may be useful in ethnic studies. Although a possible pathogenic mutation was uncovered, mutations of the nonpolymorphic portions of the TAD of the AR do not appear to have a major role in the aetiology of idiopathic male infertility.     

p53-dependent impairment of T-cell proliferation in FADD dominant-negative transgenic mice

Members of the tumour necrosis factor (TNF) receptor family exert pleiotropic effects and can trigger both apoptosis and proliferation [1]. In their cytoplasmic region, some of these receptors share a conserved sequence motif - the 'death domain' - which is required for transduction of the apoptotic signal by recruiting other death-domain-containing adaptor molecules like the Fas-associated protein FADD/MORT1 or the TNF receptor-associated protein TRADD [2-4]. FADD links the receptor signal to the activation of the caspase family of cysteine proteases [5,6]. Functional inactivation of individual receptor family members often fails to exhibit a distinctive phenotype, probably because of redundancy [7-9]. To circumvent this problem, we used a dominant-negative mutant of FADD (FADD-DN) which should block all TNF receptor family members that use FADD as an adaptor. We established transgenic mice expressing FADD-DN under the influence of the lck promoter and investigated the consequences of its expression in T cells. As expected, FADD-DN thymocytes were protected from death induced by CD95 (Fas/Apo1), whereas apoptosis induced by ultraviolet (UV) irradiation, anti-CD3 antibody treatment or dexamethasone was unaffected, as was spontaneous cell death. Surprisingly, however, we also observed profound inhibition of thymocyte proliferation in vivo and of activation-induced proliferation of thymocytes and mature T cells in vitro. This inhibition of proliferation was not due to increased cell death and appeared to be p53 dependent.     

Ulcerative colitis with overexpression of p53 preceding overt histological abnormalities of the epithelium

Laser-induced fluorescence of pheophorbide a (Ph-a) was used for in vitro photodynamic imaging (PDI) of a rat pancreatic acinar tumor. A 400 nm excitation induced a 470 nm autofluorescence and a 678 nm dye fluorescence in tumors and their surrounding pancreas 24 h after a 9 mg kg-1 body weight Ph-a intravenous administration. With lower intensities in these blood-rich tumors than in pancreas, Ph-a fluorescence signals are unable to provide tumor images. A dimensionless function (the ratio of Ph-a fluorescence by autofluorescence, called Rt for the tumor and Rp for the pancreas) was used for fluorescence contrast calculation (C = Rt/Rp) between six tumors and their paired pancreas. Among five available laser excitation wave-lengths, only the 355 nm excitation gave a distinctive contrast (C = 1.5). The PDI of six intrapancreatic tumors and their intraperitoneal metastasis and of two control normal pancreas was thus performed ex vivo using a 355 nm excitation source delivered by a tripled Nd:YAG laser and a charged-coupled device camera. Fluorescence images were recorded at 680 nm (dye), 640 nm (background) and 470 nm (autofluorescence) through three corresponding 10 nm width bandpass filters. Computed division for each pixel of Ph-a fluorescence values by autofluorescence generated false color image. In this way, contrasted tumor images were obtained. But in five out of six animals false-positive images were present due to an autofluorescence decrease in some normal pancreatic areas. A 470 nm autofluorescence imaging on the same tumors gave in all cases false-positive image and false-negative in half of the cases. These observations suggest that autofluorescence alone is unable to achieve accurate PDI of pancreatic carcinoma and that using Ph-a as a PDI dye needs strong improvements.