Allelic deletion fingerprinting of urine cell sediments in bladder cancer.

BACKGROUND: Bladder cancer shows frequent nonrandom allelic deletion at various chromosomal regions. Genotypic detection methods could potentially identify patients at risk for recurrent progressive disease. In this study, we examined allelic deletion at specific chromosomal loci in tumor tissue and urine cell sediment samples using a microsatellite-based protocol. Although both allelic deletion and microsatellite instability have been reported in primary bladder cancer, microsatellite instability was not specifically examined in this study. We report a pilot study of 40 patients with bladder cancer in which allelic deletion in tumor tissue and urine cell sediment was compared with conventional urine cytology results. METHODS AND RESULTS: Forty tumors were analyzed using a set of microsatellite primers from chromosomes 3, 4, 8, 11, 14, and 17 to construct allelic deletion fingerprints. Cy5.5-labeled PCR products were analyzed using the OpenGene System and GeneObjects software. Eighty-eight percent of tumors showed allelic deletion. In urine cell sediments, the tumor detection rate was 80% compared with 50% for routine urine cytology. The allelic deletion fingerprinting (ADF) procedure identified 69% of incipient tumors, cases initially classified as normal by routine urine cytology. CONCLUSION: ADF analysis provides a reliable noninvasive method for the detection and monitoring of recurrent cancer in urine cell sediment samples from patients with bladder cancer.

Cyclosporine challenge test revisited: does it predict outcome after solitary pancreas transplantation?

BACKGROUND: The selection of patients for solitary pancreas transplantation (PTA) requires identification of individuals who will not develop acute renal dysfunction in response to immunosuppressants. A cyclosporine challenge test (CCT) was developed to predict post-PTA kidney dysfunction secondary to calcineurin inhibitor immunosuppressants. We now report on the long-term follow-up of patients who received a PTA after undergoing a CCT. METHODS: Twelve potential PTA recipients were administered cyclosporine A (CsA) for 6 wk. Creatinine clearance (CrCl) was measured at 2, 4, and 6 wk. Those who did not fail the CCT received PTA. Baseline and post-transplant CrCl were retrospectively evaluated in the original cohort and in a group of matched patients who received PTA without a CCT. RESULTS: Of the original 12 recipients evaluated with the CCT, 6 received PTA. CrCl was followed for a mean of 45.8 months. Of the 4 who remained alive, 2 went on to develop renal failure (CrCl < 30 mL/min) at 18 and 65 months post-transplant. The baseline CrCl was higher in PTA recipients who had not been selected to be studied with CCT than those that were (117 +/- 32 vs 78 +/- 13 mL/min). By 12 months post-PTA, the CrCl was no longer different between the groups selected to be screened with CCT and those that were not. CONCLUSIONS: CCT may help predict risk for short-term changes in renal function (< 18 months) in response to CsA. CCT may be most helpful in candidates for PTA with borderline renal insufficiency (60-80 mL/min).

Patterns of p53 gene mutations in head and neck cancer: full-length gene sequencing and results of primary radiotherapy.

p53 gene alterations are common in head and neck cancers, but their prognostic value has not been clearly established. Despite evidence in other cancers that sequencing of the entire p53 coding region provides prognostic information, full-length p53 gene sequencing has rarely been performed in head and neck cancers. In this study, p53 was assessed in a series of 42 pretreatment biopsies from patients with laryngeal carcinomas by full-length gene sequencing and by immunohistochemistry (IHC). Associations among p53 genotype, protein expression, and local recurrence were assessed in 35 irradiated patients followed for a minimum of 5 years. DNA was extracted from formalin-fixed, paraffin-embedded biopsies, and exons 2-11 of the p53 gene were individually amplified by PCR and then directly sequenced. IHC was performed to detect mutant and wild-type p53 protein using the DO7 monoclonal antibody. p21 protein expression was assessed using the EA1 monoclonal antibody. Twenty genetic alterations were observed in 42 tumors (48%). Four of these alterations (20%) occurred outside exons 5-8. There was a significant association between p53 gene and protein status (chi2 = 4.18, P = 0.04), although the correlation was weak (phi coefficient = -0.327). Although local relapse following radiation was significantly associated with nodal status, no correlations were observed between p53 status (gene or IHC) and local recurrence following radiation therapy, based on the Kaplan-Meier method. These results show that p53 mutations are common in laryngeal carcinomas and that a proportion occur outside traditionally examined regions. The lack of correlation between p53 status and local control suggests that this marker is not as powerful as traditional prognostic factors, such as lymph node status.

Exon 5 of the p53 gene is a target for deletions in ovarian cancer.

Missense point mutations, leading to inactivation of the p53 tumor suppressor gene product, are currently the most frequent alterations in human cancer. Little, however, is known about small intragenic deletions or insertions occurring in this locus of chromosome 17. We have analyzed 56 primary ovarian tumors for the presence of such abnormalities. The analysis was based on multiplex PCR amplification of exons 1 through 11 of the p53 gene and fragment analysis of the generated PCR products. Mutations were detected in 14% (8 of 56) of the tumors. Deletions were much more prevalent than insertions (seven vs one). Six of the deletions and the insertion affected exon 5, and the other deletion was in exon 7. Two deletions and the insertion did not disrupt the reading frame; the protein product was expressed in the tumor at high concentrations in all three cases. The other five deletions generated a frameshift, which is predicted to result in the production of a truncated protein product. In the case of the deletions, a 2-5-bp repeat was present close to the detected deletion, whereas the insertion duplicated the sequence immediately upstream of the insertion site. Overall our findings indicate that small intragenic p53 deletions/insertions are not rare events in ovarian cancer, and that p53 exon 5 is the target in the vast majority (88%) of the cases.

Allelic deletion at chromosome 11p13 defines a tumour suppressor region between the catalase gene and D11S935 in human non-small cell lung carcinoma.

A previous study of 48 primary non-small cell lung carcinomas (NSCLC) for allelic loss at five polymorphic chromosome 11p loci indicated regions of allelic deletion for both 11p13 and 11p15. To further delineate the extent of this deletion 28 NSCLCs were examined by high resolution deletion mapping for allelic loss at 11p13.95% (18/28) of the informative cases displayed allelic loss at the catalase gene locus (CAT), 64% (18/28) at D11S935 and D11S941E, respectively, and 23% (6/26) at D11S907. The minimal region of deletion is bordered by CAT and D11S935 and spans about 3 cM. The relationship between allelic loss within this chromosomal region, the presence of a putative predisposition locus and the pathogenetics of NSCLC are discussed.

p53 protein accumulation and p53 gene alterations (RFLP, VNTR and p53 gene mutations) in non-invasive versus invasive human transitional bladder cancer.

Eleven non-invasive and 24 invasive transitional cell bladder cancers were analysed for molecular alterations to the p53 gene and nuclear accumulation of the p53 protein. 9% (1/11) of non-invasive rumours and 21% (5/24) of invasive tumours revealed nuclear accumulation in more than 50% of the tumour cells. PCR analysis of D17S30 showed loss of heterozygosity (LOH) in invasive tumours (3/24; 12%). Two invasive tumours harboured point mutations in exon 6 and exon 7, respectively (8%). Our results indicate that p53 protein overexpression correlates with tumour progression, p53 gene mutations and LOH detected by PCR.

Frequent TP53 gene alterations (mutation, allelic loss, nuclear accumulation) in primary non-small cell lung cancer.

Mutations of the TP53 tumour suppressor gene have been reported for many human cancers. A variety of TP53 mutations have also been reported for both primary non-small cell lung cancer (NSCLC) and associated metastases. To assess the pathogenetic significance of TP53 gene alterations in NSCLC, 24 paired samples of primary NSCLC and the corresponding normal lung tissue were analysed for mutations of the TP53 gene (exons 5-8) using exon-specific PCR, single-strand conformation polymorphism PCR (SSCP-PCR) and direct DNA sequencing; for p53 protein accumulation by immunohistochemistry and for 17p allelic loss using restriction fragment length polymorphism (RFLP) probes on Southern blots and amplified fragment length polymorphism-PCR. TP53 point mutations were observed in 9/24 (38%) tumours encompassing a total of 14 mutations. Two tumours displayed the same double mutation while a third harboured four different mutations. Seventeen of 24 NSCLCs (71%) overexpressed p53 protein and all 17 immunopositive tumours (100%) showed a mutation and/or allelic loss at the D17S30 locus. Of the 17 NSCLCs informative at the DS17S30 locus, 10 (59%) showed allelic loss, of which five (50%) were also mutated on the remaining TP53 allele. These results suggest that TP53 gene alterations are involved in the pathogenesis of primary NSCLC and that such alterations may serve a selective role in the development of NSCLC by diminishing the apoptotic potential of bronchial epithelial cells heterozygous for a TP53 point mutation. This may also explain the accumulation of multiple TP53 point mutations in 3/24 of our NSCLC samples.

Tyrosine kinase-dependent and -independent events induced by interleukin-2 stimulation: interleukin-2-mediated NO production required for the induction of lymphokine-activated killer cell activity in rat splenocytes is tyrosine kinase independent.

Nitric oxide (NO) has recently been shown to be an indispensable co-factor in the generation of lymphokine-activated killer (LAK) cells induced by interleukin-2 (IL-2). Upon stimulation with IL-2, cells endowed with specific receptors undergo phosphorylation of substrates mediated by protein tyrosine kinases (PTK). In this work we utilized a well-characterized PTK inhibitor, genistein (GEN), to address the role of PTK on NO-dependent LAK cell generation. The effects of GEN were tested on the expression of the inducible NO synthase (iNOS) gene, proliferation, generation of cytotoxic activity and production of NO upon IL-2 stimulation of rat splenocytes. We report here that GEN displays profound inhibitory effects on recombinant (r)IL-2 induced proliferation and on LAK cell generation, while only marginally affecting NO production, measured as NO2-. In contrast, a specific inhibitor of the NO synthetic pathway (NG-monomethyl-L-arginine; NMMA) blocked generation of LAK cells and NO production without affecting cell proliferation. If added directly to the cytotoxicity tests, GEN exerted minor inhibitory effects, not exceeding 25% of control tests, while NMMA was completely ineffective. Sodium nitroprusside (SNP), a non-enzymatic NO-releasing substance, restored LAK cell generation in cultures performed in the presence of NMMA, but not in those performed in the presence of GEN. These results indicate that IL-2-induced NO production is a PTK-independent event. IL-2-stimulated LAK cell generation obligatorily requires the concurrent activation of PTK dependent and independent signal transduction pathways.

Identification of genes differentially expressed in normal lung and non-small cell lung carcinoma tissue.

Using a magnet-assisted subtraction technique, 17 complementary DNA (cDNA) clones were isolated that were expressed in the normal lung but were decreased or lost in the corresponding tumor tissue of a nonsmall cell lung carcinoma patient. The lack of expression of six magnet-assisted subtraction technique cDNA clones in three additional non-small cell lung carcinoma cases indicates their possible relevance for non-small cell lung carcinoma. Two cDNA clones revealed homology to genes specifically expressed in lung, i.e., pulmonary surfactant-associated protein B and the receptor for advanced glycosylation end products of proteins. Three cDNA clones showed identity to cDNA sequences encoding calmodulin-like protein, glutamine synthetase, and cytoskeletal beta-actin. One cDNA clone is identical to a recently described human expressed sequence tag whose gene is still unknown.

Multistep transformation in low-grade lymphoproliferative diseases.

BACKGROUND: Carcinogenesis, the formation of solid tumors, is now widely accepted to represent a multistep process. Several genetic events, activation of proto-oncogenes and inactivation of tumor suppressor genes, are involved. DESIGN: Review of the literature for evidence that the concept of multistep transformation has relevance also for the formation of low-grade lymphoproliferative diseases. RESULTS AND CONCLUSION: The common translocations in low-grade lymphoid tumors are probably early events, predominantly involved in the activation of oncogenes, leading to growth stimulation or prolonged cell survival. As a result 'monoclonal lymphoproliferative disorders of undetermined significance (MLDUS)' occur, undetermined, because some translocations may not always led to tumor formation. For progression to full malignancy, additional genetic events are required besides sequential selection of variant subpopulations within the neoplastic clone. Recent data indicate that mutations and deletions of putative tumor suppressor genes, including the P53 and retinoblastoma genes, are also involved in the progression of lymphoproliferative disorders. A list of lymphoproliferative diseases stressing this concept of multistep transformation is presented in this article.

The Wilms tumour gene WT1 is expressed in murine mesoderm-derived tissues and mutated in a human mesothelioma.

The tumour suppressor gene WT1 encodes a transcription factor expressed in tissues of the genito-urinary system. Inactivation of this gene is associated with the development of Wilms tumour a pediatric kidney cancer. We show that WT1 is also expressed at high levels in many supportive structures of mesodermal origin in the mouse. We also describe a case of adult human mesothelioma, a tumour derived from the peritoneal lining, that contains a homozygous point mutation within WT1. This mutation, within the putative transactivation domain, converts the protein from a transcriptional repressor of its target sequence to a transcriptional activator. The role of WT1 in normal development thus extends to diverse structures derived from embryonic mesoderm and disruption of WT1 function contributes to the onset of adult, as well as pediatric, tumours.

Loss of heterozygosity on chromosome 11p13 in primary bladder carcinoma.

Although the occurrence of bladder cancer is common, the molecular events underlying the pathogenesis of this cancer remain ill-defined. A loss of heterozygosity (LOH) at specific chromosomal loci may predispose individuals to the development of bladder cancer but this has not been examined in detail. Furthermore, the role that deletion or inactivation of putative tumour suppressor genes might play in the genesis of bladder cancer has not been established. In this study, allelic deletion analysis on the short arm of chromosome 17 of patients with primary bladder tumours failed to show deletion at 17p13 (0/7), a region known to contain the p53 tumour suppressor gene. Chromosome 11p15 showed allelic deletion at the IGF2 locus (2/7: 29%) and the PTH locus (1/11: 9%). However, no deletion was observed at the CALCA locus (0/6). LOH at 11p13, a region containing the Wilm's tumour suppressor gene (WT1), was also studied. Analysis of LOH at 11p13 showed deletion at the CAT locus (13/18: 72%), the delta J/D11S414 locus (5/15: 33%), the WT1 locus (7/14: 50%) and the FSHB locus (6/16: 38%). The significance of these findings is discussed.

Expression of the novel intermediate filament-associated protein restin in Hodgkin's disease and anaplastic large-cell lymphoma.

In this study, the expression of the novel intermediate filament protein Restin in human tissues was analyzed. Restin expression was studied by immunohistochemistry using polyclonal and monoclonal antibodies. Restin was not detected in normal tissues, a range of B- and T-cell non-Hodgkin's lymphomas, and nonlymphoid tumors. However, Restin was present in Reed-Sternberg cells and variants thereof in Hodgkin's disease, with the exception of the lymphocyte-predominant, paragranuloma subtype. Restin was also highly expressed in anaplastic large-cell lymphoma (so-called Ki-1 lymphoma). As expected, Restin was also expressed in Hodgkin cell lines L428, L428KSA, Co, and KM-H2 and the anaplastic large-cell lymphoma cell line Karpas 299, which was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting, as well as Northern blotting. The presence of Restin in both Hodgkin's disease and anaplastic large-cell lymphoma is intriguing and might indicate a role of this structural protein in the pathogenesis of both conditions.

The replicative restriction of lymphocytotropic isolates of HIV-1 in macrophages is overcome by TGF-beta.

In vitro exposure of human blood monocyte-derived macrophages to T-cell tropic human immunodeficiency virus (HIV) isolates fails to establish a productive viral infection. Several studies have shown that such preferential HIV-1 replication in T cells or in mononuclear phagocytes (HIV tropism) may be determined by distinct viral characteristics. In the present study it was demonstrated that transforming growth factor-beta (TGF-beta), a factor known to be produced by platelets, macrophages, and other cells present at a wound site, can act as a mediator in overcoming the lymphocytotropic restriction of several well-characterized viral isolates of HIV-1 (i.e., LAV, Z84, pLAI, NY5). Macrophages infected with these isolates show cytopathic changes comparable to those seen upon infection with the monocytotropic isolate ADA. To achieve this effect with TGF-beta, the factor must be present after the infection period. The emerging virus retains its original cellular tropism. Based on these observations the authors propose a role for TGF-beta in the establishment and progression of HIV infection and disease.

In vitro effect of transforming growth factor-beta on progression of HIV-1 infection in primary mononuclear phagocytes.

In vitro-differentiated monocytes can be infected with the monocytotropic isolate of HIV-1/ADA. The infection is characterized by formation of giant cells and production of virus that can be found in cell supernatants or cell-associated. In this study, we demonstrate that the above described parameters of infection can be enhanced by a factor present in acidified M phi supernatants, suggesting that it might be transforming growth factor beta-1 (TGF-beta 1). When recombinant or purified TGF-beta were examined, similar activities were detected. This effect apparently is not because of changes in the cellular phenotype that could favor infection. The effect of TGF-beta is exerted on cells once infection is established or on cells with active virus production. The activity can be also demonstrated using U-937 cells.

TGF-beta: upregulator of HIV replication in macrophages.

TGF-beta at physiological concentrations, when added to monocyte-derived macrophages following HIV1 infection, has an enhancing effect upon the rate of virus production. This effect is observed with the monocytotropic isolate ADA, as well as with HIV1 IIIB, which poorly replicates in macrophages.

Expression of a leukemia-associated antigen (CAMAL) in four myeloid leukemia cell lines.

We have previously described the detection and partial characterization of a common myelogenous leukemia-associated antigen (CAMAL), in CGL and ANLL patients. Both polyclonal and monoclonal (CAMAL-1) antibodies have been raised to p70 (CAMAL) and have been shown to react with both p70 and myeloid leukemia cell preparations. p70 (CAMAL) has been shown to be a monomeric protein of Mr 70,000 and pI 7.2 and was also detectable in the myeloid leukemia cell lines HL60, KG1, K562 and U937, but not in the lymphocytic cell lines Molt-4, Hut-78 and CEM by immunoprecipitation from iodinated cell samples. Using [35S] methionine-labeled cell lines and immunoprecipitation, we have demonstrated the constitutive expression of p70 as well as a major component at p58 and a number at lower molecular weights in the myeloid leukemia cell lines HL60, KG1, K562 and U937, but not in the lymphocytic leukemia cell lines Molt-4, Hut-78 and CEM. The implications of these observations are discussed.

The use of dietary fiber in the management of simple, childhood, idiopathic, recurrent, abdominal pain. Results in a prospective, double-blind, randomized, controlled trial.

Recurrent abdominal pain (RAP) affects 10% to 18% of school-age children and is caused by obvious organic pathology in fewer than 10% of cases. Two recent studies do not support previous beliefs that most RAP is psychogenic. Studies have shown disorders of bowel motility in children with RAP similar to those of adult irritable bowel syndrome (IBS); controlled trials of additional dietary fiber in adult IBS have shown beneficial results. We did a randomized, double-blind, placebo-controlled study in 52 children with RAP and demonstrated a clinically and statistically significant decrease in pain attacks (at least 50% fewer) in almost twice as many children who were given additional fiber as placebo. Compliance was excellent in both groups and side effects were few. Although the cause of RAP is poorly understood, it is hypothesized that the beneficial effect of added fiber is due to its effect on shortening transit time, as in IBS.