PTEN and PIK3CA gene copy numbers and poor outcomes in non-small cell lung cancer patients with gefitinib therapy.

METHODS: Fluorescent in situ hybridisation analyses of PTEN, PIK3CA, EGFR and CEN7 were performed on tumour specimens from patients treated on the expanded access gefitinib trial. Progression-free survival (PFS) and overall survival (OS) were correlated with outcomes in all patients and EGFR wild-type patients. RESULTS: Progression-free survival (hazard ratio=2.54, P<0.001) and OS (hazard ratio=4.04, P<0.001) were significantly shorter in patients whose tumours had all of the following molecular patterns: CEN7 <4 copies per cell, PTEN loss (<2 copies in at least 20% of cells), and PIK3CA gain (>2 copies in at least 40% of cells) both in all and EGFR wild-type only patients. CONCLUSION: The combination of low CEN7 copy number, PTEN loss, and PI3KCA gain may be useful for identifying NSCLC patients unlikely to benefit from treatment with EGFR (TKIs), specifically in wild-type EGFR cases.

Genomic integration of oncogenic HPV and gain of the human telomerase gene TERC at 3q26 are strongly associated events in the progression of uterine cervical dysplasia to invasive cancer.

Recently proposed events associated with the progression of cervical intraepithelial neoplasia (CIN) 2/3 to cervical carcinoma include integration of human papillomavirus (HPV) into the host genome, genomic instability, and an increase in chromosome 3q copy number. In particular, the gene coding for the RNA component of telomerase (TERC) at 3q26 has been implicated as a possible candidate gene. Since it is not known to date how these events are temporally related during cervical carcinogenesis, the aim of the present study was to assess the correlation between TERC gene copy number and the physical status of HPV during progression in cervical neoplasia. Solitary precursor lesions of the uterine cervix (CIN 2/3, n = 17), lesions associated with a micro-invasive carcinoma (CIN 3&mCA, n = 13), and advanced invasive carcinomas (invCA, n = 7) were analysed by fluorescence in situ hybridization (FISH) to determine the physical status of the virus and TERC gene copy number. The TERC gene was increasingly gained with progression of CIN 2/3 (3 of 17) through CIN 3&mCA (7 of 13) to invCA (5 of 7). In the lesions exhibiting gain of TERC, the virus was predominantly integrated. This was seen in eight of ten diploid lesions, indicating that these events can occur prior to aneuploidization and are strongly associated with the progression of CIN 3 to mCA and invCA (p < 0.001). With progression to carcinoma, a number of these lesions show polyploidization, resulting in aneuploidy and high TERC gene copy numbers. In conclusion, genomic integration of oncogenic HPV and gain of the human telomerase gene TERC appear to be important associated genetic events in the progression of uterine cervical dysplasia to invasive cancer.

Frequent gain of the human telomerase gene TERC at 3q26 in cervical adenocarcinomas.

The level of genomic amplification of the human telomerase gene TERC, which maps to chromosome band 3q26, was determined in primary cervical adenocarcinomas. Interphase nuclei prepared from archival material of 12 primary cervical adenocarcinomas, eight of which were human papillomavirus positive, were hybridised with a triple colour probe set specific for centromeres of chromosomes 3 and 7 and the TERC gene. We observed high proportions of nuclei with increased absolute copy numbers for TERC in all tumours (mean 3.3; range 2.3-5.2). Amplification of the human telomerase gene TERC is a consistent aberration in cervical adenocarcinomas. Therefore, application of our probe set may provide an objective genetic test for the assessment of glandular cells in Pap smears and hence for the diagnosis of cervical adenocarcinomas.

Asymptomatic primary Epstein-Barr virus infection occurs in the absence of blood T-cell repertoire perturbations despite high levels of systemic viral load.

Primary infection with the human herpesvirus, Epstein-Barr virus (EBV), may result in subclinical seroconversion or may appear as infectious mononucleosis (IM), a lymphoproliferative disease of variable severity. Why primary infection manifests differently between patients is unknown, and, given the difficulties in identifying donors undergoing silent seroconversion, little information has been reported. However, a longstanding assumption has been held that IM represents an exaggerated form of the virologic and immunologic events of asymptomatic infection. T-cell receptor (TCR) repertoires of a unique cohort of subclinically infected patients undergoing silent infection were studied, and the results highlight a fundamental difference between the 2 forms of infection. In contrast to the massive T-cell expansions mobilized during the acute symptomatic phase of IM, asymptomatic donors largely maintain homeostatic T-cell control and peripheral blood repertoire diversity. This disparity cannot simply be linked to severity or spread of the infection because high levels of EBV DNA were found in the blood from both types of acute infection. The results suggest that large expansions of T cells within the blood during IM may not always be associated with the control of primary EBV infection and that they may represent an overreaction that exacerbates disease.

Suppression of a mitochondrial tRNA gene mutation phenotype associated with changes in the nuclear background.

We previously have characterized a pathogenic mtDNA mutation in the tRNAAsn gene. This mutation (G5703A) was associated with a severe mitochondrial protein synthesis defect and a reduction in steady-state levels of tRNAAsn. We now show that, although transmitochondrial cybrids harboring homoplasmic levels of the mutation do not survive in galactose medium, several galactose-resistant clones could be obtained. These cell lines had restored oxidative phosphorylation function and 2-fold higher steady-state levels of tRNAAsn when compared with the parental mutant cell line. The revertant lines contained apparently homoplasmic levels of the mutation and no other detectable alteration in the tRNAAsn gene. To investigate the origin of the suppression, we transferred mtDNA from the revertants (143B/206 TK-) to a different nuclear background (143B/207 TK-, 8AGr). These new transmitochondrial cybrids became defective once again in oxidative phosphorylation and regained galactose sensitivity. However, galactose-resistant clones could also be obtained by growing the 8AGr transmitochondrial cybrids under selection. Because the original rate of reversion was higher than that expected by a classic second site nuclear mutation, and because of the aneuploid features of these cell lines, we searched for the presence of chromosomal alterations that could be associated with the revertant phenotype. These studies, however, did not reveal any gross changes. Our results suggest that modulation of the dosage or expression of unknown nuclear-coded factor(s) can compensate for a pathogenic mitochondrial tRNA gene mutation, suggesting new strategies for therapeutic intervention.

Two-color ratio-coding of chromosome targets in fluorescence in situ hybridization: quantitative analysis and reproducibility.

Ratio coding in fluorescence in situ hybridizations has the potential to identify more DNA and RNA targets simultaneously using fewer fluorescent labels than other multi-color techniques. Ratio coding uses hybridization probes containing different proportions of two or more distinguishable labels to stain each target. In order to better define the limits of ratio coding applied to chromosome detection, we have 1) examined methods of processing electronic images of ratio-coded hybridizations to increase our ability to visually and quantitatively distinguish different stained targets and 2) examined the reproducibility of target identification. A number of hybridizations were performed using eight ratios of two fluorescent labels to identify whole chromosomes in metaphase spreads, and using five ratios of two labels on repetitive sequence probes to identify chromosomes in metaphase spreads and interphase nuclei. Greater visual discrimination was afforded by color composite images which expanded the color range across the entire visual spectrum. Quantitative ratio measurements on 25 metaphase spreads predicted that eight different chromosomes can be identified simultaneously with 96% accuracy using whole chromosome probes. Analysis of a smaller data set predicted that six different chromosomes can be identified with 97% accuracy using repetitive sequence probes.

Fluorescence in situ hybridization: powerful molecular tool for cancer prognosis.

We review several aspects of fluorescence in situ hybridization (FISH) technology that demonstrate its breadth and power in detecting and monitoring genetic abnormalities associated with cancers. The clinical utility of FISH in disease management is demonstrated in several examples, including trisomy 8 detection with high specificity and sensitivity in patients with myeloid leukemias; trisomy 12 detection with higher efficiency than conventional cytogenetics in patients with chronic lymphocytic leukemia; assessment of engraftment success, chimerism, and relapse in opposite sex bone marrow transplantation; and correlation of trisomy 7 with survival time in patients with prostate tumors. Advances in FISH technology include multicolor analyses, which permit the simultaneous detection of several genetic abnormalities by using cohybridization of probes labeled with several fluorescent labels or label combinations, and comparative genomic hybridization, a relatively new method whereby a single hybridization can reveal aberrations across the entire genome.

The role of the carboxy terminus of v-Rel in transformation and activation of endogenous gene expression.

Proteins within the Rel/NF-kappa B transcription factor family can be divided into two functional domains, a homologous amino terminal region, the Rel Homology Domain, and a divergent carboxy terminal domain. The amino terminal sequences specify DNA binding, nuclear localization, and interaction with the I kappa B family of inhibitory proteins. The carboxy terminus of each protein functions as a transcriptional activation domain, however, precise definition of sequence requirements has been difficult. To further define these sequences, small 100 bp deletions were constructed throughout the carboxy terminus of v-Rel. Each resulting mutant was assayed for DNA binding, localization, protein complex formation, activation of endogenous gene expression and ability to transform bone marrow cells and fibroblasts. Surprisingly, deletion within the carboxy terminus had marginal effects on transforming potential. However, three separate regions were required for full activation of gene expression. Taken together, these results suggest that the carboxy terminus of v-Rel contains multiple sequences that participate in the activation of gene expression.

Sensitive fluorescence-based thermodynamic and kinetic measurements of DNA hybridization in solution.

Kinetic and thermodynamic constants associated with DNA hybridization were determined in solution using fluorescence measurements and complementary fluorophore-labeled oligomers. One oligomer was labeled with a 5'-terminal fluorescein, and the other was labeled with a 3'-terminal rhodamine. The juxtaposition of the two labels in double-stranded complexes results in a strong quenching of the fluorescein emission, thereby providing the means for distinguishing single-stranded DNA from double-stranded DNA. Since measurements were based on fluorescence, DNA denaturation and association could be monitored routinely at strand concentrations 100-1000-fold lower than permitted by absorbance hypochromicity measurements. To determine if fluorescence quenching mirrored base pair formation, temperature profiles of DNA association and dissociation were constructed from both absorbance hypochromicity and fluorescence quenching measurements at a number of different DNA concentrations. Analyses of these profiles using the "all-or-none" model of hybridization provided thermodynamic data which were statistically indistinguishable between the two measurement methods, thus validating the use of fluorescence quenching in thermodynamic studies of oligomers. The effects of fluorophore attachment on the thermodynamic properties of the DNA strands were investigated by analyzing the melting curves of different combinations of unlabeled and labeled complementary oligomers. The presence of both labels was found to stabilize the double-stranded DNA by about -1.5 kcal in delta G degrees 298, primarily due to the fluorescein label. Association and dissociation rate constants were determined by fluorescence measurements at different temperatures, and linear Arrhenius plots were obtained. The fluorescence measurements provided a unique "label dilution" method for measuring dissociation rate constants of oligomers based upon the dynamic association and dissociation of complementary DNA strands at constant temperature. Association rate measurements were simplified since relatively low concentrations of complementary oligomers could be mixed, thereby reducing hybridization rates and eliminating the need for rapid mixing and measurement techniques.

Mutations in the rel-homology domain alter the biochemical properties of v-rel and render it transformation defective in chicken embryo fibroblasts.

Sequential deletions of approximately 100 base pairs were made in the rel-homology domain of the viral rel protein. Each deletion mutant was cloned into a replication-competent viral vector and assayed in chicken embryo fibroblasts (CEFs). The deleted v-rel proteins were analysed for localization, complex formation and ability to induce transformation. In vitro-translated mutant proteins were assayed for binding to a NF-kappa B consensus sequence. All the deletion mutants between nucleotides 37 and 798 in v-rel were transformation defective. Each of the mutants localized predominantly in the cytoplasm, whereas wild-type v-rel localizes predominantly in the nucleus of CEFs. Any disruption of the rel-homology domain reduced binding of the mutant v-rel proteins to the cellular protein, p36, while the requirements for binding to p68c-rel, p115 and p124 appeared to be more complicated. The binding of these three proteins to v-rel appeared to be linked and mediated through c-rel, suggesting that v-rel disrupts normal c-rel function. None of the deletion mutants in this region were able to bind to the NF-kappa B site. However, mutants which lie outside the rel-homology domain retained the ability to transform CEFs, localize to the nucleus, complex with p36, p115, p124 and p68c-rel and bind to the NK-kappa B site. These results suggest that transformation by v-rel requires an intact rel-homology domain and that the biochemical properties of v-rel are linked and dependent upon higher order protein structure for full function.

Expression of v-rel in a replication competent virus: transformation and biochemical characterization.

The avian reticuloendotheliosis virus strain T (REV-T) transforms bone marrow cells and may cause phenotypic changes in fibroblasts. Both events are thought to result from expression of the v-rel oncoprotein, a member of the NF-kappa B family of transcription factors. Most REV stocks contain a cytopathic and immunosuppressive helper virus (REV-A) unrelated to standard avian retroviruses, and thus the degree to which v-rel expression alone contributes to the transformed phenotype in bone marrow cells and fibroblasts is complicated by helper virus expression. To gain a more accurate picture of how v-rel contributes to transformation, we have cloned the v-rel gene into a replication-competent avian retrovirus vector (RCAS) and have expressed it in both chick embryo fibroblasts (CEF) and bone marrow cells. Transfection of RCAS-rel into CEF readily produced a partially transformed phenotype, demonstrating that expression of the v-rel protein is sufficient for fibroblast transformation. The RCAS-rel virus also transformed bone marrow cells in vitro, but required culture conditions different from those normally required for transformation by REV-T. The v-rel protein expressed in transformed CEF was biochemically indistinguishable from that expressed in transformed bone marrow cells, being localized to the cytoplasm and the nucleus, and forming a complex with cellular proteins. We also demonstrate that the RCAS-rel-transformed hematopoietic cells exhibited a distinct differentiation phenotype.

Solution-phase detection of polynucleotides using interacting fluorescent labels and competitive hybridization.

DNA was assayed in a homogeneous format using DNA probes containing hybridization-sensitive labels. The DNA probes were prepared from complementary DNA strands in which one strand was covalently labeled on the 5'-terminus with fluorescein and the complementary strand was covalently labeled on the 3'-terminus with a quencher of fluorescein emission, either pyrenebutyrate or sulforhodamine 101. Probes prepared in this manner were able to detect unlabeled target DNA by competitive hybridization producing fluorescence signals which increased with increasing target DNA concentration. A single pair of complementary probes detected target DNA at a concentration of approximately 0.1 nM in 10 min or about 10 pM in 20-30 min. Detection of a 4 pM concentration of target DNA was demonstrated in 6 h using multiple probe pairs. The major limiting factors were background fluorescence and hybridization rates. Continuous monitoring of fluorescence during competitive hybridization allowed correction for variable sample backgrounds at probe concentrations down to 20 pM; however, the time required for complete hybridization increased to greater than 1 h at probe concentrations below 0.1 nM. A promising application for this technology is the rapid detection of amplified polynucleotides. Detection of 96,000 target DNA molecules in a 50-microliters sample was demonstrated following in vitro amplification using the polymerase chain reaction technique.

Viral rel and cellular rel associate with cellular proteins in transformed and normal cells.

The rel oncogene from the avian reticuloendotheliosis virus strain T is a 59 kd phosphoprotein localized primarily to the cytoplasm of transformed cells. Recently, the v-rel protein was shown to associate with several cellular proteins with molecular weights of 124 kd, 115 kd, and 36 kd. We have analysed the subcellular distribution of v-rel protein complexes after biochemical fractionation of [35S]methionine and [32P]orthophosphate labeled cells. Our results demonstrate that the v-rel protein coprecipitates with a characteristic set of proteins, some of which are distinct to nuclear or cytoplasmic fractions. We also demonstrate that the normal cellular homolog of the viral rel protein, c-rel, coprecipitates with several cellular proteins from normal chick hematopoietic tissue. These cellular proteins have apparent molecular weights similar to those which are coprecipitated with v-rel from cytoplasmic fractions. Our results demonstrate that both v-rel and c-rel interact with a variety of cellular proteins and suggest that this association is important for the function or regulation of the rel protein.

Time-resolved detection of energy transfer: theory and application to immunoassays.

Energy-transfer measurements based upon acceptor fluorophore emission are plagued with background fluorescence resulting from absorption of the excitation light by the acceptor fluorophore. The present work examines the use of a long-lifetime donor fluorophore and a short-lifetime acceptor fluorophore, combined with pulsed-laser excitation and electronic gating of detector signals, to separate the component of acceptor emission due to energy transfer from the component due to absorption of the excitation light. Theoretical equations describing the acceptor fluorescence and integrated acceptor fluorescence show that increasing the integration delay relative to the excitation pulse should greatly enhance detection of the energy-transfer component. The time-resolved detection of energy transfer was tested in a competitive immunoassay format in which antibodies to human immunoglobulin G (IgG) F(ab')2 fragments were covalently labeled with pyrenebutyrate (tau = 100 ns) and IgG Fab' fragments were covalently labeled with B-phycoerythrin (tau = 2.5 ns). Solutions containing these two conjugates exhibited energy transfer from the pyrenebutyrate to the B-phycoerythrin upon excitation with a nitrogen laser. Acceptor emission was measured with 0- and 20-ns integration delays and the ratios of the energy-transfer component to the laser-excited component were found to increase by 9- to 15-fold when the 20-ns delay was used in three series of immunoassays. Good agreement between the experimental data and theory was obtained following convolution of the theoretical fluorescence responses with the instrumental response of the fluorometer.

Biological membrane modeling with a liquid/liquid interface. Probing mobility and environment with total internal reflection excited fluorescence.

Total internal reflection of exciting light, in combination with fluorescence intensity and polarization measurements, was used to selectively study fluorescent compounds adsorbed to the interface region between two immiscible liquids. A fluorometer was constructed which provided excitation at variable angles of incidence and allowed sensitive detection of polarized fluorescence emitted from the interface. The compound 4,4'-bis-1-phenylamino-8-naphthalenesulfonate (bis-ANS) was examined at a decalin/water interface and was found to possess remarkable affinity for the interface region with the bulk of the adsorbed molecule residing in the decalin phase. The adsorbed fluorophore displayed an apparent hindered rotation in the plane of the interface with a rotational diffusion coefficient 3- to 12-fold lower than that expected for bis-ANS in solution. While other dyes examined were not found to be significantly surface active, the addition of cationic surfactant sufficed to induce adsorption of the anionic fluorophore 1-aminonaphthalene-3,6,8-trisulfonic acid. This fluoropore was found to reside in an aqueous environment when bound to the interface, and it also exhibited hindered rotation in the plane of the interface. As the concentrations of the dyes were increased, both adsorbed dyes exhibited polarization reductions consistent with excitation energy transfer. Adsorption of bis-ANS was reversed by addition of bovine serum albumin. The membrane protein cytochrome b5 was found not to bind at the decalin/water interface, indicating that interaction with lipid is required for its adherence to biological membranes.

DNA damage and selective toxicity of dopa and ascorbate:copper in human melanoma cells.

Six of eight human melanoma lines showed increased sensitivity to killing by dopa and by ascorbate:copper compared with two fibroblast strains and four other human cell lines of nonmelanoma origin. Catechol, epinephrine, and alpha-methyldopa, but not 5,6-dihydroxyindole, exhibited a similar degree of selectivity. Toxicity was greatly reduced when brief exposure times or high cell densities were used. Depending upon culture conditions, melanoma cells accumulated more [3H]dopa- and [14C]ascorbate-derived isotopic label within the first five min than fibroblasts, but after one hr this difference was less marked. The catalase activity in melanoma cells was not less than that in fibroblasts. Using two independent methods to determine each type of damage, dopa and ascorbate:copper were found to induce DNA breaks in both cell types but not DNA repair synthesis or DNA interstrand cross-links. More DNA breaks were found in melanoma cells (two lines) than in fibroblasts. Semiconservative DNA synthesis was inhibited immediately, recovered within six hr, and in melanoma cells, was again inhibited after 24 hr. RNA synthesis was inhibited less than DNA synthesis. Human cell lines with differential sensitivity to gamma-radiation, ultraviolet light, cross-linking agents, or monofunctional alkylating agents, exhibited normal survival levels when treated with dopa or ascorbate:copper.