Neuroprotective potential of pleiotrophin overexpression in the striatonigral pathway compared with overexpression in both the striatonigral and nigrostriatal pathways.

Intrastriatal injection of recombinant adeno-associated viral vector serotype 2/1 (rAAV2/1) to overexpress the neurotrophic factor pleiotrophin (PTN) provides neuroprotection for tyrosine hydroxylase immunoreactive (THir) neurons in the substantia nigra pars compacta (SNpc), increases THir neurite density in the striatum (ST) and reverses functional deficits in forepaw use following 6-hydroxydopamine (6-OHDA) toxic insult. Glial cell line-derived neurotrophic factor (GDNF) gene transfer studies suggest that optimal neuroprotection is dependent on the site of nigrostriatal overexpression. The present study was conducted to determine whether enhanced neuroprotection could be accomplished via simultaneous rAAV2/1 PTN injections into the ST and SN compared with ST injections alone. Rats were unilaterally injected in the ST alone or injected in both the ST and SN with rAAV2/1 expressing either PTN or control vector. Four weeks later, all rats received intrastriatal injections of 6-OHDA. Rats were euthanized 6 or 16 weeks relative to 6-OHDA injection. A novel selective total enumeration method to estimate nigral THir neuron survival was validated to maintain the accuracy of stereological assessment. Long-term nigrostriatal neuroprotection and functional benefits were only observed in rats in which rAAV2/1 PTN was injected into the ST alone. Results suggest that superior preservation of the nigrostriatal system is provided by PTN overexpression delivered to the ST and restricted to the ST and SN pars reticulata and is not improved with overexpression of PTN within SNpc neurons.

p53-deficient mice are extremely susceptible to radiation-induced tumorigenesis.

Mice constitutively lacking alleles of the p53 tumour suppressor gene spontaneously develop lymphomas and sarcomas. We report here that a single dose of 4 Gy radiation dramatically decreases the latency for tumour development in p53 heterozygous mice. The pattern of genetic alterations at the remaining wild type allele in these tumours differs substantially from spontaneous tumours from similar mice indicating that p53 itself may have been a target for radiation-induced alterations. Lower dose irradiation (1 Gy) of preweanling p53 null mice also significantly decreases tumour latency, suggesting that there are additional genetic targets involved in radiation-induced malignancy. Thus p53-deficient mice provide a sensitive model system for studies of the consequences of radiation exposure.

Distinct genetic loci control development of benign and malignant skin tumours in mice.

Genetic susceptibility to chemically induced skin cancer in mice is controlled by multiple unlinked genetic loci. Mus spretus mice have dominant resistance genes which confer resistance to interspecific F1 hybrids with susceptible Mus musculus strains. We have mapped three major resistance loci using a combination of Mapmaker/QTL analysis and multiple regression analysis to mouse chromosomes 5 and 7. At least two independent loci on chromosome 7 exert their effects primarily during benign tumour development and have very little influence on tumour progression. On the other hand, probably a single locus on chromosome 5 affects both early and late stages of malignancy. The results indicate that benign and malignant tumours are largely under independent genetic control.

Synthetic lethality between mutation in Atm and DNA-PK(cs) during murine embryogenesis.

The gene product mutated in ataxia telangiectasia, ATM, is a ubiquitously expressed 370 kDa protein kinase that is a key mediator of the cellular response to DNA damage [1]. ATM-deficient cells are radiosensitive and show impaired cell cycle arrest and increased chromosome breaks in response to ionizing radiation. ATM is a member of the phosphatidylinositol-3-kinase (PI3K)-related protein kinase superfamily, which includes the catalytic subunit of DNA-dependent protein kinase (DNA-PK(cs)) and ATR [2]. DNA-PK is a 470 kDa protein kinase that is required for proper end-to-end rejoining of DNA double-strand breaks [3]. Prkdc(scid/scid) mice have a homozygous mutation in the gene encoding DNA-PK(cs) and, like Atm(-/-) mice, are viable and radiosensitive [4-8]. To determine if Atm and DNA-PK(cs) show genetic interaction, we attempted to generate mice deficient in both gene products. However, no scid/scid Atm(-/-) pups were recovered from scid/scid Atm(+/-) intercrosses. Developmental arrest of scid/scid Atm(-/-) embryos occurred around E7.5, a developmental stage when embryonic cells are hypersensitive to DNA damage [9]. This reveals synthetic lethality between mutations in Atm and DNA-PK and suggests that Atm and DNA-PK have complementary functions that are essential for development.

Genetic variation in liver tumor susceptibility, plasma testosterone levels, and androgen receptor binding in six inbred strains of mice.

We compared six inbred mouse strains for their relative susceptibilities to liver and lung tumor induction. Male and female mice were treated at 12 days of age with a single i.p. injection of N-ethyl-N-nitrosourea (ENU; 0.25 mumol/g), and tumor multiplicity was analyzed at 32 weeks of age (males) or 44 weeks of age (females). Male mice of the SWR/J and C57BL/6J strains were relatively resistant to hepatocarcinogenesis, averaging 0 and 0.3 tumors per animal, respectively. Male C57BR/cdJ, P/J, and SM/J mice had intermediate susceptibilities, averaging seven to 17 tumors per animal, and male CBA/J mice were the most susceptible, averaging 45 tumors per animal. Female mice were more resistant than male mice: no liver tumors were observed for SWR/J females; C57BL/6J, SM/J, P/J, and CBA/J females averaged less than one tumor per animal and C57BR/cdJ females averaged five tumors per animal. In contrast to the results for liver tumor induction, there was no difference between the sexes in lung tumor susceptibility. Male and female SWR/J mice were the most susceptible, averaging 14 lung tumors per animal; male and female CBA/J mice were moderately susceptible, averaging six tumors per animal and the C57BR/cdJ, C57BL/6J, P/J, and SM/J strains were relatively resistant, averaging less than three tumors per animal. To determine if levels of testosterone, a potent liver tumor promoter in mice, or its receptor contribute to the strain variation in liver tumor susceptibility, we measured levels of plasma testosterone as well as binding properties of the hepatic androgen receptor for the six inbred strains. Plasma testosterone in male mice ranged from 1.8 to 7.4 ng/ml and in females ranged from 0.21 to 0.42 ng/ml, which is consistent with the greater susceptibility of male mice to liver tumor development. However, variation in testosterone levels among the different strains of mice was not correlated with liver tumor susceptibility. We also demonstrated the presence of high affinity androgen receptors in mouse hepatic cytosol. The amounts of this receptor for the six strains tested ranged from 24 to 34 fmol/mg cytosolic protein. The apparent KD of the receptor for [3H]mibolerone (a synthetic androgen) differed between the strains: SWR/J, C57BL/6J, and C57BR/cdJ mice had the highest affinity (KD = 0.22 nM), P/J and CBA/J strains had an intermediate affinity (KD = 0.36 nM), and the SM/J strain had the lowest affinity receptor (KD = 0.45 nM). The strain variation in the affinity or abundance of the androgen receptor was not related to the strain variation in liver tumor induction.

Allelotype analysis of mouse skin tumors using polymorphic microsatellites: sequential genetic alterations on chromosomes 6, 7, and 11.

Allelotype analysis of human tumors has been instrumental in the effort to discover and clone novel tumor suppressor genes. However, this approach has not been systematically applied to animal models of carcinogenesis. We describe here the first attempt to allelotype a nonhuman tumor, i.e., chemically induced mouse skin tumors, using a panel of polymorphic microsatellite markers. The results indicated that markers on chromosomes 6 and 7 were imbalanced, consistent with trisomy in both benign and malignant skin tumors. A proportion of carcinomas also showed loss of heterozygosity on chromosome 11, where the p53 gene is located, and more rarely, on chromosomes 4, 6, and 15. The significance of these alterations is highlighted by the observations of no allelic imbalance for markers on 12 other chromosomes.

p53 induction and apoptosis in response to radio- and chemotherapy in vivo is tumor-type-dependent.

The p53 protein rapidly accumulates in cells in response to DNA damage, which can trigger apoptosis. This pathway is hypothesized to be important for tumor suppression by p53, as well as for the response of tumors to chemo- or radiotherapy. Implicit in these ideas is that the p53 induction-apoptosis pathway is active in tumor cells in vivo. Because tumor suppression by p53 in mice is markedly tissue-type-dependent, we tested the activity of the pathway in tumors in vivo by inducing tumors in six different tissues and treating tumor-bearing mice with DNA damaging cancer therapeutic agents. In response to treatment, cells from T-cell lymphomas, intestinal adenomas, and mammary tumors rapidly induced p53 and underwent apoptosis. In squamous cell papillomas, p53 was constitutively expressed and was further induced by the treatments, but apoptotic cells were only rarely observed. In treated mice bearing lung or liver adenomas, minimal or no p53 accumulation or apoptosis was observed in the tumor cells. Thus, there is marked variation in the intrinsic ability of autochthonous tumor cells to accumulate p53 and undergo apoptosis. This variation provides one explanation for the tissue specificity of tumor suppression by p53. It also indicates that the role of apoptosis in the response of tumors to therapy varies significantly among tumor types.

DNA double-strand breaks, p53, and apoptosis during lymphomagenesis in scid/scid mice.

The tumor-suppressing phenotype of p53 is thought to be due to its accumulation in response to DNA damage and resultant cell cycle arrest or apoptosis. scid/scid mice are defective in DNA double-strand break repair due to a mutation in DNA-dependent protein kinase (DNAPK). Treatment of scid/scid mice with gamma radiation or N-ethyl-N-nitrosourea resulted in approximately 86% incidence of T-cell lymphomas, compared with <6% in wild-type mice. The incidence of other tumor types was not increased in scid/scid mice, suggesting that the types of DNA double-strand break that are unrepaired in these mice are not strongly carcinogenic. To determine whether mutations in DNAPK and p53 interact, we examined mice deficient in both genes. Both scid/scid p53-/- and scid/scid p53+/- mice spontaneously developed lymphomas at shorter latency than did mice with either defect alone. Loss of the wild-type p53 allele was observed in 100% of tumors from scid/scid p53 +/- mice, indicating strong selection against p53. In contrast, p53 was not inactivated in lymphomas from scid/scid p53+/+ mice. Exposure of these tumor-bearing mice to gamma radiation resulted in p53 protein accumulation and high levels of apoptosis in all tumors that were not observed in tumors from scid/scid p53+/- mice. Thus, there was a bifurcation of molecular pathways to tumorigenesis. When p53 was heterozygous in the germ line, loss of the wild-type allele occurred, and the tumors became apoptosis resistant. When p53 was wild type in the germ line, p53 was not inactivated, and the tumors remained highly apoptosis sensitive.

Lack of transforming growth factor-beta 1 expression in benign skin tumors of p53null mice is prognostic for a high risk of malignant conversion.

Expression of transforming growth factor beta 1 (TGF beta 1) protein was examined in chemically induced benign skin tumors with genetically defined empirical risks for malignant conversion. Benign tumors induced in mice which have both alleles of the p53 gene deleted have a malignant conversion frequency of approximately 50%, whereas similar tumors induced in wildtype and heterozygous p53 mice have conversion probabilities of 3 and 8%, respectively (Kemp et al., Cell, 74: 813-822, 1993). The TGF beta 1 antibody, anti-CC (1-30-1), was shown to stain either the proliferative keratinocyte compartment of the tumor or the tumor stroma, whereas another TGF beta 1 antibody, anti-LC (1-30-1), stained highly differentiated granular cells of the tumors. A strong correlation was found between staining of the proliferative keratinocyte compartment of tumors with the anti-CC (1-30-1) antibody and tumor genotype. Only 18% (6 of 32) of homozygous p53 null tumors showed any basal keratinocyte staining with this antibody, whereas over 80% (32 of 38) of heterozygous and wild-type tumors showed positive staining. Additionally, in most tumors examined, the spatial distribution of staining for the proliferating cell nuclear antigen appeared to be mutually exclusive with that of TGF beta 1 on adjacent serial sections. This suggests that, in these cases, tumor keratinocytes are sensitive to negative growth regulation by TGF beta. TGF beta 1 protein staining in benign tumors is thus prognostic for a low probability of malignant conversion, and its expression may be mechanistically involved in limiting malignant conversion since, at the benign tumor stage examined, keratinocytes are still sensitive to growth inhibition by TGF beta 1.

Spontaneous and ionizing radiation-induced chromosomal abnormalities in p53-deficient mice.

Chromosomal abnormalities have been assessed in p53-deficient mice. The in vivo frequency of spontaneous stable aberrations in bone marrow cells was elevated by approximately 20-fold in p53 nulls and 13-fold in p53 heterozygotes compared to wild-type. No excessive induction of stable aberrations by gamma-irradiation was observed, but p53 deficiency resulted in excess radiation-induced hyperploidy (> 10-fold wild-type frequency). No influence of p53 genotype on sister chromatid exchange or G2 chromatid damage was observed in mitogen-stimulated spleen cell cultures; however, a p53 effect on postirradiation mitotic entry was seen. Abnormalities in chromosome segregation and mitotic delay following irradiation in p53-deficient mice suggest a G2-M checkpoint role for p53 and are broadly consistent with data on tumorigenesis in these animals.

The role of p53 in spontaneous and radiation-induced apoptosis in the gastrointestinal tract of normal and p53-deficient mice.

Three h after whole-body irradiation (8 Gy) of C57BL x DBA/2 F1 mice, p53 protein was expressed strongly in the stem cell compartment of the small intestine but at lower levels in the colon. At this time, apoptotic cells were also observed in the stem cell position of the small intestine, with fewer in the colon. In mice without copies of the p53 gene (nulls), the levels of spontaneous apoptosis, in both the small intestine and the colon, were not different from wild-type. Irradiation of the nulls with 8 Gy of gamma-rays failed to induce any further apoptosis: the loss of p53 essentially rendered the epithelial cells, from both the small intestine and the colon, radioresistant. The response of the epithelial stem cells of the small intestine suggests that p53 may play a role in the deletion of damaged cells with carcinogenic potential, whereas this process is limited in the colon.

Tumor suppression by p27Kip1 and p21Cip1 during chemically induced skin carcinogenesis.

p27Kip1 and p21Cip1 are cyclin dependent kinase inhibitors which can arrest cell proliferation and p27 is a tumor suppressor gene. To address the mechanism of tumor suppression by p27 and to determine if p21 has a tumor suppressor phenotype, we utilized the two stage skin carcinogenesis model on p27 and p21 knockout mice. In this model, initiation, which involves mutation of H-ras induced by DMBA, can be distinguished from promotion induced by TPA, and progression to carcinoma. The mean number of papillomas did not differ between p27-/- and control littermates, but papilloma growth rate was increased and carcinomas developed earlier. Thus, p27 deficiency did not enhance initiation, but resulted in more rapid clonal expansion of initiated cells during promotion. TPA treatment reduced p27 expression in keratinocytes also supporting a role for p27 during promotion. Tumors from p27-/- mice contained mutant H-ras indicating that p27 deficiency did not substitute for mutant ras and further, that during ras driven tumor growth, p27 is partially antagonistic since its removal led to faster growth. The treated p27-/- mice also developed intestinal adenomas. p21-/- mice did not display a significant increase in tumor numbers, growth rate or progression to carcinomas and these tumors also had mutated H-ras. Carcinomas from p21-/- mice were more poorly differentiated with a high frequency of anaplastic spindle cell carcinomas. Thus p21 deficiency mainly resulted in higher grade undifferentiated tumors.

Loss of heterozygosity and mutational alterations of the p53 gene in skin tumours of interspecific hybrid mice.

Functional alterations or loss of tumor-suppressor genes are an important feature of neoplastic progression in humans. The employment of suitable animal model systems would greatly facilitate the detection and manipulation of such genes. We describe here an experimental approach to this problem based on the analysis of skin tumors induced in F1 hybrids between Mus musculus and Mus spretus mice. The results show that loss of heterozygosity on chromosome 11 occurred in 4/13 mouse skin carcinomas, but not in premalignant papillomas. Since the murine p53 gene is located on this chromosome, immunoprecipitation and DNA-sequencing studies were carried out on tumorigenic cell lines and primary tumor DNA respectively to determine the status of p53 alleles. These studies revealed the presence of p53 mutations, both frameshifts and missense, some of which are identical to those found in human tumors. Loss of normal p53 function is found in well-differentiated squamous-cell carcinomas and thus does not appear to be directly responsible for further progression to an undifferentiated spindle cell phenotype.

Bax is required for increased enterocyte apoptosis after massive small bowel resection.

BACKGROUND: Massive small bowel resection (SBR) increases rates of both enterocyte proliferation and apoptosis. Previous studies have demonstrated increased intestinal expression of proapoptotic bax mRNA and protein, as well as the appearance of an 18-kd bax cleavage product within 12 hours of SBR. This study tested the hypothesis that bax is required for postresection increases in enterocyte apoptosis. METHODS: Male bax-null and C57Bl/6 (control) mice underwent either a 50% proximal SBR or sham operation. After 3 days, the remnant ileum was harvested and weighed. Apoptotic indexes, proliferation indexes, villus heights, and crypt depths were determined. RESULTS: The usual adaptive increases in ileal wet weight, crypt depth, and rate of proliferation occurred in both the control and bax-null mice. Resection significantly increased the rate of apoptosis in the control mice; however, it failed to alter the apoptotic index in the bax-null mice. CONCLUSIONS: Bax is necessary for the increase in apoptosis that occurs after SBR, but its absence has no significant effect on short-term adaptation. These findings suggest that enterocyte proliferation and apoptosis are differentially regulated during intestinal adaptation.

Effect of massive small bowel resection on the Bax/Bcl-w ratio and enterocyte apoptosis.

Following small bowel resection (SBR), the remnant intestine undergoes adaptation. Enterocyte proliferation is increased and counterbalanced by increased rates of apoptosis. To elucidate a mechanism for increased enterocyte apoptosis, this study tested the hypothesis that the ratio between pro-apoptotic Bax and pro-survival Bcl-w correlates with the apoptosis that occurs following SBR. Mice (C57Bl/6; n = 76) underwent a 50% proximal SBR or sham operation. After 12 hours and 1, 2, 3, and 7 days, the ileum was removed, the apoptotic index (apoptotic bodies/crypt) was recorded, and the messenger RNA and protein for Bax and Bcl-w were quantified. The apoptotic index was equivalent in the sham and SBR mice at 12 hours; however, it was significantly elevated following SBR at every other day measured. The ratio of Bax to Bcl-w messenger RNA relative to sham operation increased after SBR at 24 hours, decreased by day 3, and returned to baseline levels by 1 week. The protein ratio showed an increase by day 1, which remained elevated through day 7. An augmented ratio of Bax to Bcl-w messenger RNA and protein corresponded with the increase in enterocyte apoptosis. Alterations in the expression ratio of these genes may play a role in establishing a new homeostatic set point between proliferation and apoptosis during adaptation.

Genetic determinants of hepatocarcinogenesis in the B6C3F1 mouse.

The B6C3F1 mouse is highly susceptible to the induction of liver tumors because of the contribution of a specific gene, an allele of the Hcs (Hepatocarcinogen sensitivity) locus, inherited from its C3H inbred parent. This gene affects the rate of growth of preneoplastic hepatic lesions and results in the more rapid appearance of hepatic neoplasms in mice carrying the C3H allele in comparison to mice homozygous for the resistant C57BL/6 allele. The Hcs locus also acts synergistically with at least one class of chemical tumor promoters, the halogenated aromatic hydrocarbons. Because of this genetic promotion of hepatocarcinogenesis, B6C3F1 mice are more sensitive to liver tumor induction by both genotoxic and non-genotoxic carcinogens.

Analysis of intestinal adaptation gene expression by cDNA expression arrays.

BACKGROUND: As a tool for determining gene expression on a genomic scale, cDNA microarrays are a promising new technology that can be applied to the study of complex physiologic processes. The objective of this study was to characterize the expression of individual genes and patterns of gene expression that might provide insight into the mechanism of intestinal adaptation after massive small bowel resection. METHODS: Male ICR mice underwent a 50% proximal small bowel resection (SBR) or sham operation. After 3 days, the remnant ileum was harvested, weighed, and RNA extracted. Changes in gene expression were detected utilizing Clontech Atlas mouse cDNA expression arrays. Some of these changes were confirmed by reverse transcriptase-polymerase chain reactions (RT-PCR) and Northern blots. RESULTS: Analysis of these cDNA arrays revealed changes in the expression of multiple genes, including those involved in cell cycle regulation, apoptosis, DNA synthesis, and transcriptional regulation. The patterns of expression were consistent with the increased cell proliferation and apoptosis observed during intestinal adaptation. A large number of genes not previously associated with intestinal adaptation were identified. CONCLUSIONS: This technology may facilitate the elucidation of the intricate cellular mechanisms underlying intestinal adaptation.

Intestinal adaptation occurs independent of transforming growth factor-alpha.

BACKGROUND/PURPOSE: Signal transduction via the epidermal growth factor receptor (EGFR) is critical for intestinal adaptation after massive small bowel resection (SBR). Although it has been assumed that the major ligand for the EGFR during adaptation is EGF, the role for transforming growth factor-alpha (TGF-alpha), another major ligand for the EGFR is unknown. The purpose of this study was to test the hypothesis that TGF-alpha is an important ligand for the EGFR during intestinal adaptation. METHODS: Wild-type mice (C57BI/6) underwent a 50% proximal SBR or sham operation (bowel transection or reanastomosis) and were then assigned randomly to receive either intraperitoneal TGF-alpha or placebo. In a separate experiment, SBR or sham operations were performed in mice lacking TGF-alpha (Waved-1). After 3 days, adaptation was measured in the ileum. RESULTS: Exogenous TGF-alpha enhanced intestinal adaptation in the wild-type mice after SBR as shown by increased ileal wet weight and DNA content. Normal adaptation occurred in the mice lacking TGF-alpha as shown by increased ileal wet weight, protein and DNA content, proliferation, villus height, and crypt depth. CONCLUSIONS: Although exogenous TGF-alpha enhanced adaptation after massive SBR, adaptation was preserved in TGF-alpha-absent mice. These results refute TGF-alpha as an essential ligand for EGFR signaling during intestinal adaptation.