Mutations in the retinoblastoma-related gene RB2/p130 in primary nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is an endemic cancer in southern China and northern Africa, and its pathogenesis is not yet well defined at the molecular level. Although the involvement of p53 and of the retinoblastoma gene (RB/p105) in NPC has been well studied, there is paucity of mutational data regarding the retinoblastoma-related gene RB2/p130 in primary tumors and particularly in NPC. We have shown previously that RB2/p130 could be rearranged in a nasopharyngeal cell line. In the present study, we screened by single-strand conformation polymorphism and sequence analysis the retinoblastoma-related gene RB2/p130 for mutations within exons 19-22. Mutations in the RB2/p130 gene were detected in 3 of 10 primary human NPCs from Northern Africa (30%). These findings, along with previous data showing that genetic replacement of RB2/p130 restores a normal growth pathway in the nasopharyngeal cell line Hone-1, strengthen the hypothesis that genetic changes of RB2/p130 may be involved in the development and/or progression of nasopharyngeal cancer and suggest that RB2/p130 could be considered a tumor suppressor gene and may be a candidate for novel gene therapeutic approaches for NPC.

Genetic alterations disrupting the nuclear localization of the retinoblastoma-related gene RB2/p130 in human tumor cell lines and primary tumors.

The prototypic tumor suppressor gene, the retinoblastoma gene (RB/ p105), is mutated in a variety of human tumors. However, to date, mutational data on retinoblastoma family members p107 and RB2/p130 in tumors is lacking. We studied the expression of pRb2/p130 by immunocytochemistry and Western blot analysis in a panel of human osteosarcoma and lymphoid cell lines. Only the lymphoid cell lines showed an abnormal cytoplasmic localization of pRb2/p130, suggesting possible alterations within the region of nuclear localization signaling. We screened these cell lines for genetic alterations of the RB2/p130 gene in the region of the putative bipartite nuclear localization signal (NLS). This region is highly homologous with that of the RB/p105 gene. In addition, we screened four primary Burkitt's lymphomas for genetic alterations in the RB2/p130 gene. Naturally occurring mutations, which disrupt the putative bipartite NLS, were found in lymphoma cell lines and primary tumors, but not in the osteosarcoma cell lines, where normal nuclear localization of the protein was detectable. Site-directed mutagenesis and transfection assay using NLS mutants displayed markedly reduced biological activity as measured by flow cytometric analysis. This study clearly describes RB2/ p130 as an important target for mutations and subsequent inactivation in lymphoma pathogenesis, thus validating that RB2/p130 is a classical tumor suppressor gene.

Mutations in the retinoblastoma-related gene RB2/p130 in lung tumors and suppression of tumor growth in vivo by retrovirus-mediated gene transfer.

The retinoblastoma (Rb) family consists of the tumor suppressor pRb/p105 and related proteins p107 and pRb2/p130. Recent immunohistochemical studies of the retinoblastoma family of proteins in 235 specimens of lung cancer show the tightest inverse association between the histological grading in the most aggressive tumor types and pRb2/p130. This led us to study a panel of human lung cancers for mutations in the RB2/p130 gene. Mutations in the Rb-related gene RB2/p130 were detected in 11 of 14 (78.5%) primary lung tumors by single-strand conformation polymorphism and sequence analysis. A Moloney leukemia virus-based retroviral system was set up, and a comparable viral concentration of 1 x 10(7) infectious units/ml was obtained. Retrovirus-mediated delivery of wild-type RB2/p130 to the lung tumor cell line H23 potently inhibited tumorigenesis in vitro and in vivo, as shown by the dramatic growth arrest observed in a colony assay and the suppression of anchorage-independent growth potential and tumor formation in nude mice. The tumors transduced with the RB2/p130 retrovirus diminished in size after a single injection, and a 12-fold reduction in tumor growth after RB2/p130 transduction compared with the Pac-transduced tumors (92% reduction, P = 0.003) and lacZ-transduced tumors (93% reduction, P < 0.001) was found to be statistically significant. These findings provide the missing confirmation that RB2/p130 is a "bona fide" tumor suppressor gene and strengthen the hypothesis that it may be a candidate for cancer gene therapy for lung cancer.

A serine 37 mutation associated with two missense mutations at highly conserved regions of p53 affect pro-apoptotic genes expression in a T-lymphoblastoid drug resistant cell line.

The p53 protein accumulates rapidly through post-transcriptional mechanisms following cellular exposure to DNA damaging agents and is also activated as a transcription factor leading to growth arrest or apoptosis. Phosphorylation of p53 occurs after DNA damage thereby modulating its activity and impeding the interaction of p53 with its negative regulator oncogene Mdm2. The serines 15 and 37 present in the amino terminal region of p53 are phosphorylated by the DNA-dependent protein kinase (DNA-PK) in response to DNA damage. In order to verify if specific p53 mutations occur in the multi-drug resistance phenotype, we analysed the p53 gene in two T-lymphoblastoid cell lines, CCRF-CEM and its multi-drug-resistant clone CCRF-CEM VLB100, selected for resistance to vinblastine sulfate and cross-resistant to other cytotoxic drugs. Both cell lines showed two heterozygous mutations in the DNA binding domain at codons 175 and 248. The multi-drug resistant cell line, CCRF-CEM VLB100, showed an additional mutation that involves the serine 37 whose phosphorylation is important to modulate the protein activity in response to DNA damage. The effects of these mutations on p53 transactivation capacity were evaluated. The activity of p53 on pro-apoptotic genes expression in response to DNA damage induced by (-irradiation, was affected in the vinblastine (VLB) resistant cell line but not in CCRF-CEM sensitive cell line resulting in a much reduced apoptotic cell death of the multi-drug resistant cells.

Retinoblastoma-related p107 and pRb2/p130 proteins in malignant lymphomas: distinct mechanisms of cell growth control.

pRb/p105, p107, and pRb2/p130 compose the retinoblastoma (RB) family of proteins and regulate cellular growth and differentiation. Because recent functional studies have indicated that the expression of the RB-related proteins p107 and pRb2/p130 are tightly cell cycle regulated, we were interested in investigating their expression along with cellular kinetic characteristics and proliferative features of non-Hodgkin's lymphomas (NHLs). p107 and pRb2/ p130 expression was determined immunohistochemically in biopsy specimens from 83 untreated patients with NHLs of various histiotypes. The expression of these two RB-related proteins was correlated with the mitotic index, apoptotic index, and percentages of Ki-67(+), cyclin A(+), p34(+), and cyclin B(+) cells. The overall survival rate was evaluated according to the Kaplan-Meier method and the log-rank test. We found a positive correlation between the percentages of cells positive for p107 and proliferative features such as mitotic index and percentage of Ki-67(+) and cyclin A(+) cells, whereas such correlation could not be demonstrated for the percentages of pRb2/p130 positive cells. Low immunohistochemical levels of pRb2/p130 detected in untreated patients with NHLs of various histiotypes inversely correlated with a large fraction of cells expressing high levels of p107 and proliferation-associated proteins. Such a pattern of protein expression is normally observed in continuously cycling cells. Interestingly, such cases showed the highest survival percentage (82.5%) after the observation period of 10 years. Thus, down-regulation of the RB-related pRb2/p130 protein could be one of the reasons why these cases display such a high rate of proliferation and why they respond so well to therapy.

Multiple fluorescence and reflectance simultaneous detection by confocal microscopy of HaeIII digested DNA sequences.

Human metaphase chromosomes were isolated and digested in situ with HaeIII restriction enzyme to detect cytosine and guanine-rich sequences (CpG islands), which are known to be associated with most of the mammalian genes. Digested DNA was reconstructed by in situ nick translation employing digoxigenin-labeled nucleotides. The DNA sequences were revealed by antibodies conjugated either with fluorescein isothiocyanate or 1-nm colloidal gold. DNA was counterstained with propidium iodide. A sensitive, high resolution method for visualizing three signals, simultaneously excited by a single argon laser line of 488 nm has been developed. The green fluorescence of fluorescein isothiocyanate was detected in combination with the red fluorescence of propidium iodide, and the third signal was imaged by employing the reflectance mode of the confocal microscope after silver enhancement of the gold beads. The high reflectance intensity, the accurate localization and the non-fading properties of colloidal gold made the reaction a valuable tool for the detection of antigens and, as a consequence, of specific DNA sequences in chromosome preparations. Overlaying of three signals allowed the simultaneous observation of distinct structures: total DNA, as well as fluorescein- and gold-labeled sequences after in situ nick translation, or total DNA and centromeric sequences of two different chromosome pairs (17 and X) after in situ hybridization. The use of HaeIII restriction enzyme that cut CpG islands combined with in situ nick translation identified the chromosome sites where active, inactive or housekeeping genes can be located. In chromosomes, the fluorescent reaction pattern showed large areas of labeling, while a more defined staining, often organized in spot pairs that resembled an R-like banding, was detected when the reflected mode was used. These results are confirmed by the observation that R-like bands actually are multiple symmetrical spots localized on sister chromatids. In addition, some chromosomes, and in particular 1 and 9, displayed a C-negative banding due to the negativity of the centromeric areas. Reflectance confocal scanning microscopy and in situ nick translation represent a powerful tool to study the in situ genome organization.

Monoclonal antibodies and therapy of human cancers.

This survey is an overview of the applications of murine, humanized and recombinant monoclonal antibodies for in vivo diagnostic and therapeutic applications. Monoclonal antibodies (mAb) have been applied to the diagnosis and therapy of an array of human diseases. The initial failures of early clinical trials have been overcome through the production of a new generation of mAb which features reduced immunogenicity and improved targeting abilities. The early models of mAb therapy were focused on enhancing the cytolytic mechanisms against the tumor cells. More recently, successful mAb-based therapies were targeted to molecules involved in the regulation of growth of cancer cells. This has highlighted the relevance of understanding receptor-mediated signaling events, and may provide new opportunities for anti-tumor antibody targeting. Despite all the difficulties, clinical data is outlining an increasingly significant role for antibody-mediated cancer therapy as a versatile and powerful instrument in cancer treatment. One reasonable expectation is that treatment at an earlier stage in the disease process or in minimal residual disease may be more advantageous.

Expression of cell-cycle-associated proteins pRB2/p130 and p27kip in vulvar squamous cell carcinomas.

Squamous cell vulvar carcinoma accounts for 4% of all gynecologic malignancies. The cause of vulvar cancer is still unclear. Identification of new biologic factors involved in vulvar carcinogenesis may be useful in clarifying the natural history of this malignancy. We investigated the immunohistochemical expression of the retinoblastoma-related proteins pRB2/p130 and CKI p27kip1 in a series of 51 invasive squamous cell carcinomas of the vulva (ISCCs) and in synchronous normal vulvar skin, non-neoplastic epithelial disorders (NNED) and vulvar intraepithelial neoplasia (VIN). Normal vulvar skin staining showed positivity for both pRB2/p130 and p27kip1. Loss of pRB2/p130 occurred in 29 (57%) of 51 specimens of ISCCs, and in 1 of 7 specimens with VIN (14%; P = .04). We also observed a significant decrease of pRB2/p130 expression from NNED to neoplastic tissues (VIN and ISCCs) (P = .004). Loss of p27kip1 expression was found in 16 of 51 specimens (31%) of invasive carcinomas, in 1 (14%) of 7 specimens of VIN, and in 2 of 18 specimens of NNED (11%). pRB2/p130 and p27(kip1) did not correlate significantly with any of the clinicopathologic parameters examined. Our data indicate that loss of pRB2/p130 and p27kip1 are frequent events in invasive vulvar carcinomas compared with synchronous premalignant lesions, non-neoplastic epithelial disorders, and normal vulvar skin. The significant progressive decrease of pRB2/p130 expression from non-neoplastic epithelial alterations through intraepithelial neoplasia to invasive vulvar carcinomas suggests a role for this tumor suppressor gene in vulvar carcinogenesis.

Endothelin-1 in idiopathic pulmonary fibrosis.

AIMS--To evaluate whether endothelin-1 is involved in the pathology of idiopathic pulmonary fibrosis (IPF). METHODS--Plasma endothelin-1 concentrations were evaluated in 37 patients with IPF and 27 normal controls by radioimmunoassay. In addition, expression of endothelin-1 in lung tissue was evaluated in biopsy specimens obtained from four patients with IPF. Three biopsy specimens of normal lung were used as controls. Endothelin-1 immunoreactivity was detected using immunohistochemistry. RESULTS--Elevated endothelin-1 plasma concentrations were found in patients with IPF compared with controls and a positive correlation was found with duration of disease. No significant difference was observed between treated and untreated patients with IPF. Increased endothelin-1 immunoreactivity was found in lungs of three of four patients with IPF. Endothelin-1 positive consisted mainly of small vessel endothelial cells. Some scattered macrophages were also positive. CONCLUSIONS--Elevated plasma concentrations and expression of endothelin-1 in lung tissue are suggestive of increased production of endothelin-1 in at least a proportion of patients with IPF. Consequently, endothelin-1 activity could play a role in the fibrogenic process of the disease.

Molecular abnormalities in leukemia: the 11q23 story so far.

Various hematopoietic malignant disorders have been shown to bear chromosome abnormalities of the 11q23 band, which can be rearranged with many different chromosomal regions in a wide variety of different leukemia subtypes. Several laboratories have identified a trithorax-related gene that is involved in most of the 11q23 abnormalities. Although some patterns and associations between the partner genes are beginning to emerge, it is not yet possible to frame a single unifying hypothesis for 11q23 leukaemic transformation. The aim of this review is to summarise the recent data concerning these 11q23 rearrangements and the understanding of their consequences.

Rapid and sensitive detection of recombinant soluble proteins in the supernatant of transfected mammalian cells.

Cloning and expression of recombinant soluble proteins could be quite a difficult task, especially when it comes to reliably detect minute amounts of the soluble protein in the supernatant of transfected mammalian cells. Timing and sensitivity are of the essence in order to optimise the benefits/costs balance and to decide which clones to grow further and which ones to discard. Here we propose a modified inhibition assay. The key feature of this approach is the development of a sensitive and quantitative test to detect the presence of the recombinant soluble protein by exploiting its ability to compete with the binding of a specific monoclonal antibody to a target cell. The described procedure is a sensitive, efficient, dependable and low cost method.

How does a normal human cell become a cancer cell?

The mechanism by which cells become cancerous has been studied in several different species and cell types. Here, we will focus on the mechanism by which a normal human cell becomes a cancer cell and specifically discuss genes that researchers have used to transform cells. Studying how those genes affect cellular immortalization and transformation will help researchers understand more about cancer biology, find new treatments for cancer and/or improve cell survival during gene therapy.

Apoptotic pathways depend on the target enzymatic activity and not on the triggering agent.

Molt-4 human leukemia cells were triggered to apoptosis by various agents with different mechanisms of action. Staurosporine, a protein kinase C (PKC) inhibitor; camptothecin, a topoisomerase I blocking drug; and tiazofurin, an inhibitor of inosine 5'-phosphate dehydrogenase (IMPDH), were used. Ultrastructural analysis showed morphologic changes characteristic of apoptosis that were very similar for all three agents. Nevertheless, DNA oligonucleosomic fragmentation was not detectable by agarose gel electrophoresis. However, a genomic DNA cleavage appeared after pulse-field gel electrophoresis (PFGE) in cells treated with these agents for 24 h. Furthermore, in situ nick translation (NT) showed a finely spotted nuclear labelling in staurosporine-treated cells and a compact fluorescence after camptothecin incubation. In tiazofurin-treated cells an intermediate pattern was found. Therefore, apoptotic agents with different mechanisms of action induced the formation of large genomic DNA fragments and very similar ultrastructural changes. Therefore, both phenomena and the closely related apoptosis progression depend on target cell machinery and not on the triggering agent.

Perception of methacholine-induced airway obstruction in asthmatics.

The main objective of this study was to examine the perception of the first symptom during methacholine bronchoconstriction as soon as it occurred, and in the second instance to quantify the intensity of the breathlessness by means of the Borg Scale performed at the end of challenge so to not determine any difficulties for identification of the first symptom. A methacholine challenge test was carried out in 139 symptom-free asthmatics with a normal pulmonary function. When the first symptom was reported by the subject, the forced expiratory volume in one second (FEV1) was measured immediately. FEV1 was expressed as a percentage of the best personal value (FEV1%BPV). The intensity of the breathlessness was rated using a modified Borg Scale at the end of the challenge test so as not to confuse the patient. Seven subjects felt nothing during challenge. The first symptoms varied: constriction behind the breastbone (49%), inspiratory shortness (16.5%), coughing (10%), wheezing, throat constriction, general chest tightness, pain behind the sternum, and a sensation of rheum behind the sternum. FEV1%BPV at the first symptom was 80.5 +/- 10 (range 41-99). No symptom was perceived by 42% of the subjects within the 80-100 range of FEV1%BPV. FEV1%BPV at the first symptom was related to the log of the provocative dose causing a 20% fall in FEV1 (r = 0.2, p < 0.05). An inverse correlation between Borg Score and final FEV1%BPV (r = -0.25, p < 0.01) was found at the end of challenge. A subgroup of 39 subjects with similar final FEV1%BPV values (68-72) showed a correlation between the Borg Score at end of challenge and FEV1%BPV at the first symptom (r = 0.59, p < 0.001). The first symptom of the methacholine-induced bronchoconstriction varies in asthmatics and may be atypical, the bronchoconstriction level at which it is felt also varies among individuals, highly methacholine-responsive subjects perceive the bronchoconstriction later, while late perceivers of the first symptom show less intense breathlessness at the end of challenge. The measurement of the patient's ability to perceive asthmatic symptoms during the methacholine challenge test could be used to single out poor perceivers.

Assessment of emphysema using high resolution CT, CT expiratory density mask, and plain chest films.

Pulmonary function tests (diffusing capacity for carbon monoxide of the lungs) and radiological imaging (plain chest film, high resolution computed tomography (CT) and CT expiratory density mask) were compared in the assessment of 29 patients with suspected airways obstruction. Conventional roentgenogram showed a good agreement with the diffusing capacity of the lungs and proved to be useful in predicting the presence of severe emphysema, but the extension of the disease was more precisely assessed by computed tomography. A good agreement was found between high resolution CT and density mask CT, although the "subjective" high resolution identified more patients with mild emphysema than the "automated" density mask. In conclusion, although the plain chest film is useful in the diagnosis of severe emphysema, CT (especially when high resolution is used) is helpful in identifying cases of mild disease and in diagnosing the type of emphysema.

Application of the primer in situ DNA synthesis (PRINS) technique to titer recombinant virus and evaluation of the efficiency of viral transduction.

Titration is an important and critical step in dosing recombinant virus for gene therapy. We present a relatively fast, convenient, and sensitive method that allows for precise quantification of recombinant retrovirus. The method is based on PCR amplification of a foreign gene by the PRINS (primer in situ DNA synthesis) technique. The PRINS technique is based on the sequence-specific annealing of unlabeled oligonucleotide DNA in situ. This oligonucleotide operates as a primer for in situ chain elongation catalyzed by the Taq I polymerase. Using digoxygenin-labeled nucleotides as a substrate for chain elongation, the neo-synthetic DNA is labeled by an FITC-conjugated anti-digoxygenin antibody. To avoid the possibility of false positives, we amplified the puromycin-resistance gene, which is associated with the transgene in the same viral vector and is not normally present in mammalian cells. The retroviral titer was evaluated by counting fluorescein isothiocyanate-positive cells after PRINS labeling, while knowing the number of plated cells that were transduced with different amounts of viral supernatant. A comparable viral concentration of 1 x 10(7) infectious units/mL was found among the retroviruses.

Ultrastructural banding induced by DraI or HaeIII progressive digestion and in situ nick translation on human chromosomes.

Fixed human metaphase chromosomes were progressively digested with DraI or HaeIII restriction enzymes, submitted to in situ nick translation, and observed by transmission electron microscopy to obtain further information on the localization of the endonuclease target sequences and on the conformational changes in chromosomal bands. This approach allows us to detect specific nick translation patterns, namely, G-banding or R-like banding after short DraI and HaeIII endonuclease digestion, respectively. Intermediate banding recognizable as C-negative banding and G + C banding are induced by longer HaeIII digestion, before the C-positive banding. These patterns appear to depend both on different target sites of the employed endonucleases and on the DNA loss at different digestion times.