Differential response of tumor cells and normal fibroblasts to fractionated combined treatment with topotecan and ionizing radiation.

PURPOSE: The impact of the topoisomerase-I inhibitor topotecan (Hycamtin) as a single agent or in combination with ionizing radiation on clonogenic cell survival has been evaluated in three tumor cell lines and in normal fibroblasts. Both agents have been applied in a fractionated scheme in order to assess clinically relevant conditions. MATERIALS AND METHODS: Cell inactivation was investigated in human glioblastoma cell lines U118 and U138, lung carcinoma cell line A549 and normal fibroblasts of the skin (HSF6) and lung (CCD32) using the colony formation assay. RESULTS: The glioblastoma cell lines were highly sensitive to the drug, whereas normal fibroblasts were much less sensitive. A significant antagonistic effect of the drug combined with irradiation was found for normal fibroblasts, while glioblastoma cells showed no evidence of interaction, indicating additivity. A549 cells showed a biphasic response to topotecan alone and in combination with irradiation, suggesting induction of resistence to the drug. CONCLUSION: Differential effects of combined topotecan/radiation treatment were detected between cells of normal tissue and tumor cells, being antagonistic in fibroblasts of the skin and lung, but not in tumor tissue, especially in glioblastoma cells.

Loss of heterozygosity at 11p15 and p53 alterations in malignant gliomas.

PURPOSE: Malignant gliomas are the most frequent primary brain tumors. Recent studies defined several genetic markers, which might characterize molecular-biological subsets of glioblastomas with prognostic implications. In the later steps of tumor-progression, deletions on chromosome 11p15 and mutations of the tumor suppressor gene p53 were determined for different malignancies. To elucidate the involvement of 11p15 deletions in the tumorigenesis of malignant gliomas, we analyzed a series of 50 glioblastomas for loss of heterozygosity (LOH). METHODS: Paired tissue and blood samples from 50 patients with glioblastoma multiforme were included. Microsatellite markers located on 11p15.1-11p15.5 were used for LOH analysis. Additionally, mutation analysis of the tumor suppressor gene p53 was performed, which might correlate with favorable survival in glioblastomas. RESULTS: The region 11p15.4-5 was deleted heterozygously in 28% of cases representing 15 cM. Twenty-six glioblastomas did not show allelic loss for any locus. Our data revealed close association of LOH 11p15 with p53 mutations, and survival analysis showed a trend indicating better prognosis in glioblastomas characterized by LOH 11p15. CONCLUSION: In the tumorigenesis of malignant gliomas, p53 mutations and 11p15 deletions seem to indicate a genetic subset of tumors with favorable prognostic value.

Analysis of mdm2 and p53 gene alterations in glioblastomas and its correlation with clinical factors.

Malignant gliomas are the most frequent primary brain tumors. Recent studies defined several genetic markers, which might characterize molecular-biological subsets of glioblastomas with probably prognostic implications. To elucidate the involvement of murine-double-minute (mdm)2 gene amplifications and mutations of the tumor suppressor gene p53 in the tumorigenesis of malignant gliomas we analyzed a series of 75 glioblastomas. The p53 mutations occur in one-third of glioblastomas, mdm2 amplifications were found in 13% of cases. Our analysis revealed a hot spot in the p53 gene locus in codon 156, the same point mutation was detected in 4 tumor samples. None of the mdm2 amplified tumors had p53 mutations, supporting the hypothesis, that mdm2 amplifications are alternative mechanisms for p53 inactivation. Patients with p53 mutated tumors were significantly younger characterized by a mean age of 44 years. Additionally association with longer overall survival could be detected for this subgroup of patients. In our study, survival estimation revealed a significant correlation of mdm2 gene amplification with shorter survival time, and support the hypothesis, that mdm2 oncogene activation appears to occur late in tumor progression and may be characteristic as negative prognostic marker.

The presence of wild-type TP53 is necessary for the radioprotective effect of the Bowman-Birk proteinase inhibitor in normal fibroblasts.

In the present study we have demonstrated that the Bowman-Birk proteinase inhibitor (BBI) protected normal fibroblasts from a radiation-induced reduction in cell survival, whereas in transformed fibroblasts no radioprotective effect was observed. It was shown that BBI reduced the radiation-induced protein stabilization and DNA-binding activity of TP53 (formerly known as p53) in normal fibroblasts. In transformed fibroblasts, BBI failed to induce these effects. The analysis of the TP53 gene in transformed fibroblasts revealed a mutation in exon 5. As a consequence of this mutation, the expression of the TP53 downstream gene CDKN1A (p21/WAF1/Cip1) is blocked. Based on experiments using TP53 antisense oligonucleotides, the radioprotective effect of BBI could be correlated with the function of wild-type TP53. Thus BBI can be considered as a selective radioprotective agent for normal human fibroblasts.

High frequency of chromosome 9p allelic loss and CDKN2 tumor suppressor gene alterations in squamous cell carcinoma of the bladder.

BACKGROUND: In the Western Hemisphere, 90% of bladder cancers are transitional cell carcinomas, while only 7% are classified as squamous cell carcinomas. In contrast, in Egypt and regions of the Middle East and Africa, where infection by the trematode Schistosoma haematobium is endemic, squamous cell carcinoma is the most common bladder cancer as well as the most common cancer in men. PURPOSE: We planned experiments to understand the genetic defects underlying the development of squamous cell carcinoma and to determine if the morphologically and clinically distinct squamous cell carcinoma and transitional cell carcinoma of the bladder evolve following different genetic alterations. METHODS: Squamous cell carcinoma specimens from high-risk (Egypt, n = 19) and low-risk (Sweden, n = 12) populations were examined for genetic defects known to be involved in transitional cell carcinoma tumorigenesis. Homozygous deletions of the CDKN2 tumor suppressor gene were detected by comparative multiplex polymerase chain reaction. Mutations in the CDKN2 and p53 (also known as TP53) genes were analyzed by single-strand conformation polymorphism and DNA sequencing. Immunohistochemical staining of p53 protein was also performed. Allelic losses in chromosome arms 9p, 9q, and 17p were determined by microsatellite analysis. RESULTS: Homozygous deletions and sequence mutations in the CDKN2 gene were found in 67% (eight of 12) of squamous cell carcinoma specimens, a frequency three times higher than that reported for uncultured transitional cell carcinomas (P = .009). Hemizygous and homozygous deletions in 9p, where CDKN2 resides, were found in 92% (11 of 12) of uncultured squamous cell carcinomas, while only about 39% (35 of 90) of transitional cell carcinomas showed these losses (P = .001). Deletions in 9p with no change in 9q were found in 92% (10 of 11) of squamous cell carcinomas compared with only 10% (11 of 110) of transitional cell carcinomas (P < .001) reported in the literature. The frequency of p53 mutations in squamous cell carcinomas was similar to that reported for invasive transitional cell carcinomas (60%), but the type and position of mutations differed between the two tumor types. Allelic losses in chromosome arm 17p, where the p53 gene resides, were found to be less frequent in squamous cell carcinomas (38%) than in invasive transitional cell carcinomas (60%). CONCLUSIONS: Our results suggest that a putative tumor suppressor gene on 9p, possibly CDKN2, may contribute to squamous cell carcinoma tumorigenesis. Our data on squamous cell carcinoma and previously reported data on transitional cell carcinoma indicate that these two bladder carcinomas differ in their genetic alterations, suggesting that distinct underlying genetic defects may explain, at least in part, the pathological differences between the two tumors of the bladder epithelium. IMPLICATIONS: Development of diagnostic and therapeutic strategies for squamous cell carcinoma of the bladder based on its distinct genetic alterations is warranted.

Mutational spectrum in the p53 gene in bladder tumors from the endemic area of black foot disease in Taiwan.

An elevated risk of bladder cancer has been reported in the endemic region of 'black foot disease' on the southwest coast of Taiwan and may be related to high arsenic levels in artesian well water. Thirteen urothelial tumors from this endemic region were examined for mutations in exons 5-8 of the p53 gene to identify the effects of possible exogenous factors at the DNA level. DNA was extracted from archival tissue after microdissection of tumors and analyzed by PCR-SSCP (polymerase chain reaction-based single strand conformation polymorphism), followed by direct sequencing. Eight cases (62%) showed mutations and 9 of the 10 point mutations observed were transitions. The type and position of the mutations were not significantly different when compared with the spectra of p53 mutations previously reported for transitional cell carcinomas (TCCs). However, two of the mutations were CGC-->CAC base changes at codon 175, a mutational hotspot for many tumor types but previously unreported in TCCs except in cases associated with inflammatory agents. Three of the tumors examined were found to contain double mutations, a relatively rare mutagenic event in human cancers. Our results suggest that the agents responsible for the high risk of bladder cancer in the black foot disease region may operate through an inflammation-based mechanism which increases the amount of DNA damage per mutagenic event.

Two molecular pathways to transitional cell carcinoma of the bladder.

Noninvasive transitional cell carcinomas of the bladder can have two distinct morphologies suggesting they contain different genetic alterations. Papillary transitional cell carcinomas (T(a) tumors) are often multifocal and only occasionally progress, whereas flat tumors (carcinomas in situ, CIS), frequently progress to invasive disease. We examined 216 bladder tumors of various stages and histopathologies for two genetic alterations previously described to be of importance in bladder tumorigenesis. Loss of heterozygosity of chromosome 9 was observed in 24 of 70 (34%) T(a) tumors but was present in only 3 of 24 (12%) CIS and dysplasia lesions (P = 0.04). In contrast, only 1 of 36 (3%) T(a) tumors contained a p53 gene mutation compared to 15 of 23 (65%) CIS and dysplasias (P < 0.001), a frequency comparable to that observed in muscle invasive tumors (25 of 49; 51%). The presence of p53 mutations in CIS and dysplasia could explain their propensities to progress since these mutations are known to destabilize the genome. Analysis of several tumor pairs involving a CIS and an invasive cancer provided evidence that the chromosome 9 alteration may in some cases be involved in the progression of CIS to more invasive tumors, in addition to its role in the initiation of T(a) tumors. However, the CIS and secondary tumor were found to contain different genetic alterations in some patients suggesting divergent progression pathways. Bladder carcinogenesis may therefore proceed through two distinct genetic alteration pathways responsible for generating superficial tumors with differing morphologies and pathologies.

Distinct pattern of p53 mutations in bladder cancer: relationship to tobacco usage.

A distinct mutational spectrum for the p53 tumor suppressor gene in bladder carcinomas was established in patients with known exposures to cigarette smoke. Single-strand conformational polymorphism analysis of exons 5 through 8 of the p53 gene showed inactivating mutations in 16 of 40 (40%) bladder tumors from smokers and 13 of 40 (33%) tumors from lifetime nonsmokers. Overall, 13 of the 50 (26%) total point mutations discovered in this and previous work were G:C-->C:G transversions, a relatively rare mutational type in human tumors. In six tumors, identical AGA (Arg)-->ACA (Thr) point mutations at codon 280 were observed, suggesting a mutational hotspot in these tumors. Comparison of the mutational spectra from smokers and nonsmokers revealed no obvious differences in the types or positions of inactivating mutations; however, 5 of 15 tumors containing point mutations from cigarette smokers had double mutations, four of which were tandem mutations on the same allele. No double mutations were found in tumors from nonsmoking patients. None of the mutations in smokers were G:C-->T:A transversions, which would be anticipated for exposure to the suspected cigarette smoke carcinogen 4-aminobiphenyl. The results suggest that, although cigarette smoke exposure may not significantly alter the kinds of mutations sustained in the p53 gene, it may act to increase the extent of DNA damage per mutagenic event.

MR imaging of experimental meningeal melanomatosis in nude rats.

MR imaging of the rat brain has become an increasingly frequently used method in experimental neuroradiology. On a generally available 1.5 T whole body tomograph, supplemented with an individually made small coil and a special SE sequence we obtained fairly fine images of the structures of the rat brain. With gadolinium-DTPA, we were able to visualize posterior fossa and cervical leptomeningeal growth of intrathecally injected B16 melanoma in nude rats. Using MRI to follow experimental leptomeningeal metastasis, may provide a new means for diagnostic evaluation and preclinical testing of treatment modalities.

Intrathecal ACNU treatment of B16 melanoma leptomeningeal metastasis in a new athymic rat model.

To evaluate new cytotoxic drugs for intrathecal treatment we developed an experimental model of leptomeningeal metastasis by intracisternal injection of 10(4) B16-F10 melanoma cells in nude rats. One hour in vitro incubation with 20 micrograms/ml ACNU (area under the drug concentration-time curve = 1200 microgramsxmin/ml) induced a 4-log kill of B16 melanoma cells. A single or repeated non-toxic dose of 1 mg/kg was injected into the cisterna magna of rats inoculated with tumor (area under the drug concentration-time curve assuming an even cerebrospinal fluid distribution greater than 7000 microgramsxmin/ml). Median survival free of symptoms was 16 days (range 14-27) for controls (n = 9) and 18 days (range 17-23) for rats treated with ACNU on day 4 (n = 9). Animals treated both on day 2 and 8 (n = 8) developed symptoms on day 21 (range 13-35). Neurological symptoms and neuropathological examination in animals with increased survival indicated local suppression of tumor growth in the cisterna magna but increased spinal seeding and mass growth. From these results and the available pharmacokinetic data on ACNU it is concluded that bolus injection of ACNU--although locally effective--is not a sufficient treatment of widespread leptomeningeal metastasis. An increased therapeutic efficacy might be achieved by ventriculolumbar perfusion.