Expression-based intrinsic glioma subtypes are prognostic in low-grade gliomas of the EORTC22033-26033 clinical trial.

INTRODUCTION: The European Organisation for Research and Treatment of Cancer (EORTC) 22033-26033 clinical trial (NCT00182819) investigated whether initial temozolomide (TMZ) chemotherapy confers survival advantage compared with radiotherapy (RT) in low-grade glioma (LGG) patients. In this study, we performed gene expression profiling on tissues from this trial to identify markers associated with progression-free survival (PFS) and treatment response. METHODS: Gene expression profiling, performed on 195 samples, was used to assign tumours to one of six intrinsic glioma subtypes (IGSs; molecularly similar tumours as previously defined using unsupervised expression analysis) and to determine the composition of immune infiltrate. DNA copy number changes were determined using OncoScan arrays. RESULTS: We confirm that IGSs are prognostic in the EORTC22033-26033 clinical trial. Specific genetic changes segregate in distinct IGSs: most samples assigned to IGS-9 have IDH-mutations and 1p19q codeletion, samples assigned to IGS-17 have IDH-mutations without 1p19q codeletion and samples assigned to other intrinsic subtypes often are IDH-wildtype. A trend towards benefit from RT was observed for samples assigned to IGS-9 (hazard ratio [HR] for TMZ is 1.90, P = 0.065) but not for samples assigned to IGS-17 (HR 0.87, P = 0.62). We did not identify genes significantly associated with PFS within intrinsic subtypes, although follow-up time is limited. We also show that LGGs and glioblastomas differ in their immune infiltrate, which suggests that LGGs are less amenable to checkpoint inhibitor-type immune therapies. Gene expression analysis also allows identification of relatively rare subtypes. Indeed, one patient with a pilocytic astrocytoma was identified. CONCLUSION: IGSs are prognostic for PFS in EORTC22033-26033 clinical trial samples.

Early detection of human glioma sphere xenografts in mouse brain using diffusion MRI at 14.1 T.

Glioma models have provided important insights into human brain cancers. Among the investigative tools, MRI has allowed their characterization and diagnosis. In this study, we investigated whether diffusion MRI might be a useful technique for early detection and characterization of slow-growing and diffuse infiltrative gliomas, such as the proposed new models, LN-2669GS and LN-2540GS glioma sphere xenografts. Tumours grown in these models are not visible in conventional T2 -weighted or contrast-enhanced T1 -weighted MRI at 14.1 T. Diffusion-weighted imaging and diffusion tensor imaging protocols were optimized for contrast by exploring long diffusion times sensitive for probing the microstructural alterations induced in the normal brain by the slow infiltration of glioma sphere cells. Compared with T2 -weighted images, tumours were properly identified in their early stage of growth using diffusion MRI, and confirmed by localized proton MR spectroscopy as well as immunohistochemistry. The first evidence of tumour presence was revealed for both glioma sphere xenograft models three months after tumour implantation, while no necrosis, oedema or haemorrhage were detected either by MRI or by histology. Moreover, different values of diffusion indices, such as mean diffusivity and fractional anisotropy, were obtained in tumours grown from LN-2669GS and LN-2540GS glioma sphere lines. These observations highlighted diverse tumour microstructures for both xenograft models, which were reflected in histology. This study demonstrates the ability of diffusion MRI techniques to identify and investigate early stages of slow-growing, invasive tumours in the mouse brain, thus providing a potential imaging biomarker for early detection of tumours in humans.

WIF1 re-expression in glioblastoma inhibits migration through attenuation of non-canonical WNT signaling by downregulating the lncRNA MALAT1.

Glioblastoma is the most aggressive primary brain tumor in adults and due to the invasive nature cannot be completely removed. The WNT inhibitory factor 1 (WIF1), a secreted inhibitor of WNTs, is systematically downregulated in glioblastoma and acts as strong tumor suppressor. The aim of this study was the dissection of WIF1-associated tumor-suppressing effects mediated by canonical and non-canonical WNT signaling. We found that WIF1 besides inhibiting the canonical WNT pathway selectively downregulates the WNT/calcium pathway associated with significant reduction of p38-MAPK (p38-mitogen-activated protein kinase) phosphorylation. Knockdown of WNT5A, the only WNT ligand overexpressed in glioblastoma, phenocopied this inhibitory effect. WIF1 expression inhibited cell migration in vitro and in an orthotopic brain tumor model, in accordance with the known regulatory function of the WNT/Ca(2+) pathway on migration and invasion. In search of a mediator for this function differential gene expression profiles of WIF1-expressing cells were performed. Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), a long non-coding RNA and key positive regulator of invasion, emerged as the top downregulated gene. Indeed, knockdown of MALAT1 reduced migration in glioblastoma cells, without effect on proliferation. Hence, loss of WIF1 enhances the migratory potential of glioblastoma through WNT5A that activates the WNT/Ca(2+) pathway and MALAT1. These data suggest the involvement of canonical and non-canonical WNT pathways in glioblastoma promoting key features associated with this deadly disease, proliferation on one hand and invasion on the other. Successful targeting will require a dual strategy affecting both canonical and non-canonical WNT pathways.

p73 is not mutated in meningiomas as determined with a functional yeast assay but p73 expression increases with tumor grade.

The p53 gene is normally wild type in meningiomas. Since all three members of the p53 gene family recognize the same DNA sequence, tumors containing wild type p53 could decrease transactivation of p53 target genes by mutating either p63 or p73. In meningiomas the most likely target is p73, because loss of heterozygosity of the chromosomal band containing p73 is the commonest genetic lesion in these tumors. To screen p73 for mutations we have developed a functional assay which tests the ability of p73 to activate transcription from a p53-responsive promoter in yeast. The assay correctly identified p73 mutants with mutations equivalent to hotspot mutations in p53, demonstrating that the assay can detect transcriptionally inactive p73. No mutations in p73 were identified in meningiomas. p73 RNA level was higher in more advanced tumors, but there was no correlation between the expression level of p73 and p21, a known p53 target gene. The yeast assay was also used to measure the intrinsic sensitivity of the p73 protein to mutagenesis. Like p53, p73 is exceptionally easy to inactivate as a transcription factor by point mutation. Taken together, these results indicate that p53 and p73 serve very different functions in tumors.

Quantitative real-time PCR does not show selective targeting of p14(ARF) but concomitant inactivation of both p16(INK4A) and p14(ARF) in 105 human primary gliomas.

In many human cancers, the INK4A locus is frequently mutated by homozygous deletions. By alternative splicing this locus encodes two non-related tumor suppressor genes, p16(INK4A) and p14(ARF) (p19(ARF) in mice), which regulate cell cycle and cell survival in the retinoblastoma protein (pRb) and p53 pathways, respectively. In mice, the role of p16(INK4A) as the critical tumor suppressor gene at the INK4A locus was challenged when it was found that p19(ARF) only knock-out mice developed tumors, including gliomas. We have analysed the genetic status of the INK4A locus in 105 primary gliomas using both microsatellite mapping (MSM) and quantitative real-time PCR (QRT-PCR). Comparison of the results of the two methods revealed agreement in 67% of the tumors examined. In discordant cases, fluorescence in situ hybridization (FISH) analysis was always found to support QRT-PCR classification. Direct assessment of p14(ARF) exon 1beta, p16(INK4A) exon 1alpha and exon 2 by QRT-PCR revealed 43 (41%) homozygous and eight (7%) hemizygous deletions at the INK4A locus. In 49 (47%) gliomas, both alleles were retained. In addition, QRT-PCR, but not MSM, detected hyperploidy in five (5%) tumors. Deletion of p14(ARF) was always associated with co-deletion of p16(INK4A) and increased in frequency upon progression from low to high grade gliomas. Shorter survival was associated with homozygous deletions of INK4A in the subgroup of glioblastoma patients older than 50 years of age (P=0.025, Anova test single factor, alpha=0.05).

Reduced latency but no increased brain tumor penetrance in mice with astrocyte specific expression of a human p53 mutant.

p53-germline mutations located in the core DNA-binding domain have been associated with a more dominant tumor penetrance especially for breast cancer and brain tumors. We previously reported an unusual accumulation of CNS tumors associated with a unique p53 germline mutation, Y236delta (deletion of codon 236). To test whether this tissue-specific tumor predisposition reflects a gain-of-function activity of Y236delta, we generated transgenic mice expressing Y236delta in astrocytes using the regulatory elements of the glial fibrillary acidic protein (GFAP) gene. After transplacental exposure to N-ethyl-N-nitrosourea (25 mg/kg BW) brain tumors developed in 18% (7/39) of GFAP-Y236delta transgenic p53-/- mice, while in p53+/- mice the incidence was 28% (11/40) (P>0.3). However, the mean tumor latency for GFAP-Y236delta/p53+/- mice was significantly shorter than for p53+/- mice, with 19.9 weeks vs 31.6 weeks (P=0.039), respectively. Taken together, cell specific expression of Y236delta results in an acceleration of tumor progression but does not confer a higher tumor penetrance. Conceivably, the transdominant effect of Y236delta provided a growth advantage early in the progression of neoplastic cells, since the endogenous p53 wild-type allele was lost in all brain tumors independent of the genotype. This reflects well observations from human astrocytic neoplasms with p53 mutations.

p53 gene mutation and ink4a-arf deletion appear to be two mutually exclusive events in human glioblastoma.

P16 and P14ARF are two tumor suppressors encoded by the locus ink4a-arf which is frequently deleted in human tumors. Recent experiments performed with mouse embryonic fibroblasts have shown that P14ARF is an upstream regulator of the P53 pathway. This raises the question as to whether in human tumors the loss of p14arf and mutation of p53 are mutually exclusive events which segregate with genetic alterations at other loci. To examine this question we performed a multigenic analysis on 29 gliomas. We analysed p53 and p14arf in relation with five other genetic loci encoding the most frequently mutated genes in human gliomas: cdkn2a, mdm2, egfr, pten and the chromosomal regions 10q23.3 and 10q25-26. Our study shows for the first time that p53 mutations and p14arf deletions appear mutually exclusive in human glioblastoma, suggesting that they may be functionally redundant in glioma tumorigenesis. The P53 pathway is, therefore, disrupted in 81.8% of malignant gliomas (WHO grades III and IV), either by mutation of the p53 gene (31.8%) or by p14arf deletion (54.5%). These tumors further showed MDM2 overexpression (9.1%), egfr oncogene amplification/egfr overexpression (50%), pten mutations (27.3%) and loss of heterozygosity (LOH) at the chromosomal regions 10q23.3 (86.4%) and 10q25-26 (100%). These alterations did not segregate with p53 mutations or p14arf deletions, while p14arf and cdkn2a were always deleted.

p53 transdominance but no gain of function in mouse brain tumor model.

Although p53 mutations in tumors typically result in loss of transactivation of p53 target genes some mutants display gain-of-function activity. The latter has important implications for the design of rational cancer therapy. We previously described a germ-line p53 mutation (deletion of codon 236, Y236delta) associated with a familial brain tumor syndrome. To determine whether this tissue-specific tumor predisposition reflects a gain-of-function activity of Y236delta or an effect of genetic background we have developed a mouse brain tumor model. Primary neuroectodermal cells deficient for p53 (+/- or -/-) and transduced with Y236delta using a retroviral vector were transplanted into the brain of adult wild-type mice. This neurografting paradigm circumvents the problem of early lethal tumors at extracerebral sites associated with germ-line p53 deficiency. Brain tumors arising in this mouse model were highly invasive, reflecting an important feature of the human disease. Tumors arose from p53+/- cells only when transduced with Y236delta. In keeping with in vitro data showing that Y236delta has dominant-negative activity, these tumors retained the endogenous wild-type p53 allele but accumulated high levels of Y236delta. However, the presence of Y236delta in transplanted p53-/- cells had no effect on the tumor frequency, 15% versus 27% without the mutant. In conclusion, Y236delta is transdominant but exerts no gain-of-function activity mediating a more penetrant tumor phenotype.

No complementation between TP53 or RB-1 and v-src in astrocytomas of GFAP-v-src transgenic mice.

Human low-grade astrocytomas frequently recur and progress to states of higher malignancy. During tumor progression TP53 alterations are among the first genetic changes, while derangement of the p16/p14ARF/RB-1 system occurs later. To probe the pathogenetic significance of TP53 and RB-1 alterations, we introduced a v-src transgene driven by glial fibrillary acidic protein (GFAP) regulatory elements (which causes preneoplastic astrocytic lesions and stochastically astrocytomas of varying degrees of malignancy) into TP53+/- or RB-1+/- mice. Hemizygosity for TP53 or RB-1 did not increase the incidence or shorten the latency of astrocytic tumors in GFAP-v-src mice over a period of up to 76 weeks. Single strand conformation analysis of exons 5 to 8 of non-ablated TP53 alleles revealed altered migration patterns in only 3/16 tumors analyzed. Wild-type RB-1 alleles were retained in all RB-1+/-GFAP-v-src mice-derived astrocytic tumors analyzed, and pRb immunostaining revealed protein expression in all tumors. Conversely, the GFAP-v-src transgene did not influence the development of extraneural tumors related to TP53 or RB-1 hemizygosity. Therefore, the present study indicates that neither loss of RB-1 nor of TP53 confer a growth advantage in vivo to preneoplastic astrocytes expressing v-src, and suggests that RB-1 and TP53 belong to one single complementation group along with v-src in this transgenic model of astrocytoma development. The stochastic development of astrocytic tumors in GFAP-v-src, TP53+/- GFAP-v-src, and RB-1+/- GFAP-v-src transgenic mice indicates that additional hitherto unknown genetic lesions of astrocytes contribute to tumorigenesis, whose elucidation may prove important for our understanding of astrocytoma initiation and progression.

Functional analyses of a unique p53 germline mutant (Y236delta) associated with a familial brain tumor syndrome.

We have evaluated the functional properties of the unique p53 mutant Y236delta (deletion of codon 236) that gave rise to apparent cell-type specific tumor development. Four family members carrying this mutation in the germline developed early onset brain tumors, as previously reported. Deletion of residue Y236, which is tightly packed in an evolutionary conserved hydrophobic pocket, results in a protein with a mutant conformation according to immunoprecipitation with the conformation-sensitive antibodies PAb240 and PAb1620. The Y236delta mutant lacks specific DNA binding to the p53-responsive element in the WAF1-promoter, and functional analysis in Saos-2 cells revealed inability to transactivate the p53-responsive elements in the WAF1-promoter and the RGC sequence. The mutant has retained a functional oligomerization domain, a key element mediating the dominant negative effect, and inhibits DNA binding of wild-type p53. In addition, transactivation of endogenous wild-type p53 in LoVo cells was inhibited upon transfection of the mutant in a dose-dependent manner. Thus, in vitro and in vivo data suggest the loss of important tumor-suppressing functions and demonstrate a dominant negative effect of this unique p53 mutant that is associated with an unusual clustering of familial brain tumors.

Light-induced cell death of retinal photoreceptors in the absence of p53.

PURPOSE: Cell death by apoptosis is essential for normal development and tissue homeostasis, and it is involved also in a variety of pathologic processes. Apoptosis is the final common pathway of photoreceptor cell death in retinal dystrophies and degeneration. So far, little is known about genes regulating apoptosis in the retina. The tumor-suppressor gene product p53 is a potent regulator of apoptosis in numerous systems. However, p53-independent apoptotic pathways also have been described. In this study the authors investigated the role of p53 in the light-induced apoptosis of retinal photoreceptors using mice lacking p53. METHODS: Free-moving p53-/- and p53+/+ mice were dark adapted and were exposed to 8,500 or 15,000 lux of diffuse, cool, white fluorescent light for 2 hours. Animals were killed before and immediately after light exposure or at 12 hours in darkness after light exposure. Eyes were enucleated and processed for light and electron microscopy and histochemistry (TdT-dUTP terminal nick-end labeling method). Isolated retinas were subjected to the extraction of total retinal DNA. Electroretinogram (ERG) recordings were performed at all time points. RESULTS: Morphologic, biochemical, histochemical, and ERG analysis showed that the retinas of untreated p53-/- mice and wild-type control mice were structurally and functionally indistinguishable. After exposure to diffuse white fluorescent light, light-induced photoreceptor cell death was analyzed and was found to be the same in both groups of mice. CONCLUSIONS: These data suggest that light-induced apoptosis of photoreceptors is independent of functional p53.

Hemizygous or homozygous deletion of the chromosomal region containing the p16INK4a gene is associated with amplification of the EGF receptor gene in glioblastomas.

The p16INK4a gene product acts as a negative regulator of the cell cycle by binding to cyclin-dependent kinases (CDKs) 4 and 6, thereby inhibiting the formation of an active CDK/cyclin D complex. Deletion of the p16 locus has been observed in tumor cell lines and, less frequently, in primary human neoplasms. We analyzed 31 glioblastomas and identified 6 cases with hemizygous and 6 with homozygous deletions of the p16 locus. Eight of these cases showed a concurrent amplification of the EGFR gene (epidermal growth factor receptor) while the overall frequency was 35%. This close correlation suggests that deletion of the p16 chromosomal region constitutes another genetic hallmark of the primary glioblastoma, which rapidly develops de novo, without a less malignant precursor lesion and for which EGFR amplification is a characteristic genetic change. The p16 protein was not detectable in 15 of 22 glioblastomas but only 4 of these showed homozygous deletion of the gene. The alternative transcript p16 beta, for which a growth-suppressing function has been suggested, was co-expressed with p16 alpha mRNA in most cases. Hypermethylation of CpG islands in the 5' region of the p16 gene was identified in only 1 case, suggesting that this alternative mechanism of gene silencing is rarely responsible for loss of p16 expression in glioblastomas. Likewise, only 1 glioblastoma carried a p16 mutation and in addition, unexpectedly, a homozygous deletion of p16 in approximately 80% of tumor cells. This mutation, Arg24Pro, has previously been identified in a melanoma kindred.

Determination of p53 mutations, EGFR overexpression, and loss of p16 expression in pediatric glioblastomas.

Glioblastoma multiforme is a rare neoplasm in children and is often located infratentorially, particularly in the brainstem: Pediatric glioblastomas arise frequently (here 60%) outside the cerebral hemispheres. We investigated 20 pediatric glioblastomas for mutational inactivation of the p53 tumor suppressor gene, loss of p16 protein expression and overexpression of the epidermal growth factor receptor (EGFR). Mutations in the p53 gene were identified in 5/20 (25%) glioblastomas, 4 of which occurred in primary glioblastomas with a clinical history of less than 4 months and neither clinical nor histologic evidence of a less malignant precursor lesion. Loss of p16 expression was detected in 11/18 (61%) glioblastomas. Overexpression of the EGFR was infrequent (2/19, 11%) and included 1 tumor with a p53 mutation. Of 4 secondary glioblastomas that progressed from histologically diagnosed lower grade tumors, one contained a p53 mutation. Our results are at variance with similar studies in adult patients in which primary and secondary glioblastomas are characterized by EGFR overexpression and p53 mutations, respectively, suggesting that the evolution of pediatric glioblastomas follows different genetic pathways.

Identical mutations of the p53 tumor suppressor gene in the gliomatous and the sarcomatous components of gliosarcomas suggest a common origin from glial cells.

Gliosarcomas are morphologically heterogeneous tumors of the central nervous system composed of gliomatous and sarcomatous components. The histogenesis of the latter is still a matter of debate. As mutations of the p53 tumor suppressor gene represent an early event in the development of gliomas, we attempted to determine whether both components of gliosarcomas share identical alterations of the p53 gene. Using single-strand conformation analysis (SSCA) and direct DNA sequencing of the p53 gene, we analyzed dissected gliomatous and sarcomatous parts of 12 formalin-fixed, paraffin-embedded gliosarcomas. The two tumors that contained a p53 alteration were found to carry the identical mutation (exon 5; codon 151, CCC-->TCC; codon 173, GTG-->GTA) in the gliomatous and the sarcomatous components. These findings suggest a common origin of the two cellular components from neoplastic glial cells.

Familial brain tumour syndrome associated with a p53 germline deletion of codon 236.

This report describes clinical, neuropathological and molecular genetic findings in a Swiss family with four brain tumours in only two generations. The neoplasms observed covered a wide range of biologic behaviour, from a slowly growing lesion already apparent at birth, to anaplastic astrocytoma in a young adult and glioblastomas at the age of less than 10 years. The only non-neural neoplasms in this family were a case of leukemia and an adrenocortical carcinoma. A germline deletion of codon 236 of the p53 tumour suppressor gene was identified as an underlying cause and detected in all affected family members. This mutation has not previously been reported as germline transmission or in sporadic tumours. The unusual accumulation of CNS tumours may be due to a certain organ-specific effect of this particular p53 mutation or it may reflect the specific genetic back-ground of this family.

Allelotype analysis of mouse lung carcinomas reveals frequent allelic losses on chromosome 4 and an association between allelic imbalances on chromosome 6 and K-ras activation.

We generated allelotypes of 38 methylene chloride-induced lung carcinomas from female C57BL/6J x C3H/6J F1 (hereafter called B6C3F1) mice. Two or more polymorphic markers per autosome, most of them microsatellites, were examined for loss of heterozygosity. Allelic losses throughout the genome were generally infrequent except for markers on chromosome 4, which were lost in approximately one-half of the carcinomas. Analysis of lung adenomas indicated that chromosome 4 loss was associated with malignant conversion. In addition, chromosome 4 loss were specific for lung carcinomas based on comparison to methylene chloride-induced liver tumors and additional studies of lung tumors from a variety of treatment protocols and different mouse strains. Preferential loss of the maternal chromosome 4 was observed in B6C3F1 carcinomas. Analyses of additional tumors induced in mice from two reciprocal crosses, A/J x C3H/HeJ F1 (hereafter called AC3F1) and C3H/HeJ x A/J F1 (hereafter called C3AF1), provided evidence for the inactivation of one allele of the putative chromosome 4 tumor suppressor gene by parental imprinting. Most B6C3F1 tumors lost all chromosome 4 markers examined, suggesting nondisjunction events. In contrast, several C3AF1 and AC3F1 tumors appeared to have interstitial deletions that defined the smallest region of overlap as a 9-cM interval between Ifa-2 and D4Nds2. The homologous region on human chromosome 9p21-22 is frequently lost in a variety of tumors including lung cancers. A candidate tumor suppressor gene, MTS1, is located in this region, which is homozygously deleted or mutated in cell lines derived from a variety of human tumors. Finally, an association between K-ras gene activation and allelic imbalances on chromosome 6 was observed for B6C3F1 lung tumors.

In vitro growth characteristics of embryo fibroblasts isolated from p53-deficient mice.

Fibroblast cultures were derived from mouse embryos containing either one (p53+/-) or two (p53-/-) inactivated p53 alleles and compared to normal embryo fibroblasts for a number of growth parameters. Early passage p53-deficient embryo fibroblasts (p53-/-) divided faster than normal embryo fibroblasts, achieved higher confluent densities, and had a higher fraction of division-competent cells under conditions of low cell density. Flow cytometry studies of early passage embryo fibroblasts showed that the percent of p53-deficient cells in G0/G1 was lower than in normal cells, consistent with the argument that p53 mediates a G1 block. When p53-deficient and normal cells were passaged for long periods of time, the homozygote (p53-/-) fibroblasts grew at a high rate for over 50 passages and never entered a non-growing senescent phase characteristic of the heterozygote (p53+/-) and normal (p53+/+) cells. The p53-deficient fibroblasts were genetically unstable during passaging, with the p53-/- cells showing a high degree of aneuploidy and the p53+/- cells displaying a moderate level of chromosomal abnormalities by passage 25. Surprisingly, the heterozygote cells lost their single wild type allele very early during culturing and in spite of this loss most heterozygote lines entered into senescence. We conclude that the loss of p53 by itself is insufficient to confer immortality on a cell, but does confer a growth advantage. Taken together, the findings confirm that the absence of p53 promotes genomic instability, which in turn may result in genetic alterations which directly produce immortality.

Characterization of p53 mutations in methylene chloride-induced lung tumors from B6C3F1 mice.

Mutations of the p53 tumor suppressor gene are the most common defined genetic alterations seen in a wide variety of human cancers. In contrast, little is known about the importance of the p53 gene in chemically induced tumors of rodents, which are widely used as models for the evaluation of human health risks. In this study we examined 54 methylene chloride-induced and seven spontaneously arising lung tumors from female B6C3F1 mice for losses of heterozygosity (LOH) at markers near the p53 gene on chromosome 11. LOH was detected in seven methylene chloride-induced lung carcinomas by Southern analysis of a restriction fragment length polymorphism and PCR analysis of five simple sequence length polymorphisms. In each case allele loss was observed at all six markers; thus, these chromosomal alterations were likely to have resulted from mitotic nondisjunction. In contrast, LOH was not detected in 20 liver tumors from methylene chloride-treated mice at the Acrb locus, which is tightly linked to the p53 gene on chromosome 11. In addition single strand conformation polymorphism analysis was performed to screen for mutations in the most conserved regions of the p53 gene (exons 5 to 8). Consequently, potential mutations identified by direct sequencing, were only detected in four of the seven tumor samples with LOH, but not in any of the remaining lung tumors. Overexpression of the p53 protein by immunohistochemical staining was detected only in the four tumors that contained p53 point mutations and in a focal area of another tumor. Finally, using a simple sequence length polymorphism within the retinoblastoma tumor suppressor gene, LOH on mouse chromosome 14 was also detected in three lung carcinomas and one liver tumor. Inactivation of p53 and possibly the retinoblastoma tumor suppressor gene appear to be infrequent events in lung and liver tumors from methylene chloride treated mice.

Comparison of pulmonary O6-methylguanine DNA adduct levels and Ki-ras activation in lung tumors from resistant and susceptible mouse strains.

The role of O6-methylguanine (O6MG) DNA adduct formation and persistence in the formation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung tumors from resistant C57BL/6 and susceptible A/J mice was investigated. In addition, the frequencies of pulmonary tumor formation and Ki-ras activation were defined in C57BL/6 mice treated with NNK or vinyl carbamate (VC), and the role of the p53 gene in pulmonary carcinogenesis in these resistant mice was examined. One day after treatment with 100 mg/kg NNK, O6MG adduct concentrations were twofold to eightfold higher in Clara cells and type II cells than in small cells or whole lungs from both mouse strains. The concentrations of O6MG in isolated cells decreased at a similar rate in the two strains of mice. Lung tumors were detected by 27 mo of age in 18% of the C57BL/6 mice after a single 100 mg/kg dose of NNK and in 46% of these mice after a single 60 mg/kg dose of VC. In contrast, the tumor incidence in untreated C57BL/6 mice was 4%. Only one of 22 lung tumors from C57BL/6 mice treated with NNK contained an activated Ki-ras gene that was associated with an O6MG DNA adduct, whereas previous studies detected activated Ki-ras oncogenes in most of the NNK-induced lung tumors analyzed from susceptible A/J and resistant C3H mice. The small differences in formation and persistence of the O6MG adduct in whole lung or isolated lung cells from A/J and C57BL/6 strains do not account for the differences in either susceptibility for tumor formation or activation of the Ki-ras gene between these strains. In contrast to the low number of NNK-induced tumors with Ki-ras mutations in the resistant mice, 11 of 20 lung tumors from VC-treated mice contained activated Ki-ras genes. Neither p53 tumor suppressor gene mutations nor overexpression of the p53 protein were detected in spontaneous or chemically induced lung tumors in C57BL/6 mice. Thus, although Ki-ras activation was detected in some tumors, pathways independent of ras activation and p53 inactivation also appear to be involved in lung tumorigenesis in this resistant mouse strain.