Quantitative mapping of amplicon structure by array CGH identifies CYP24 as a candidate oncogene.

We show here that quantitative measurement of DNA copy number across amplified regions using array comparative genomic hybridization (CGH) may facilitate oncogene identification by providing precise information on the locations of both amplicon boundaries and amplification maxima. Using this analytical capability, we resolved two regions of amplification within an approximately 2-Mb region of recurrent aberration at 20q13.2 in breast cancer. The putative oncogene ZNF217 (ref. 5) mapped to one peak, and CYP24 (encoding vitamin D 24 hydroxylase), whose overexpression is likely to lead to abrogation of growth control mediated by vitamin D, mapped to the other.

PIK3CA is implicated as an oncogene in ovarian cancer.

Ovarian cancer is the leading cause of death from gynecological malignancy and the fourth leading cause of cancer death among American women, yet little is known about its molecular aetiology. Studies using comparative genomic hybridization (CGH) have revealed several regions of recurrent, abnormal, DNA sequence copy number that may encode genes involved in the genesis or progression of the disease. One region at 3q26 found to be increased in copy number in approximately 40% of ovarian and others cancers contains PIK3CA, which encodes the p110alpha catalytic subunit of phosphatidylinositol 3-kinase (PI3-kinase). The association between PIK3CA copy number and PI3-kinase activity makes PIK3CA a candidate oncogene because a broad range of cancer-related functions have been associated with PI3-kinase mediated signalling. These include proliferation, glucose transport and catabolism, cell adhesion, apoptosis, RAS signalling and oncogenic transformation. In addition, downstream effectors of PI3-kinase, AKT1 and AKT2, have been found to be amplified or activated in human tumours, including ovarian cancer. We show here that PIK3CA is frequently increased in copy number in ovarian cancers, that the increased copy number is associated with increased PIK3CA transcription, p110alpha protein expression and PI3-kinase activity and that treatment with the PI3-kinase inhibitor LY294002 decreases proliferation and increases apoptosis. Our observations suggest PIK3CA is an oncogene that has an important role in ovarian cancer.

Tumour amplified kinase STK15/BTAK induces centrosome amplification, aneuploidy and transformation.

The centrosomes are thought to maintain genomic stability through the establishment of bipolar spindles during cell division, ensuring equal segregation of replicated chromosomes to two daughter cells. Deregulated duplication and distribution of centrosomes have been implicated in chromosome segregation abnormalities, leading to aneuploidy seen in many cancer cell types. Here, we report that STK15 (also known as BTAK and aurora2), encoding a centrosome-associated kinase, is amplified and overexpressed in multiple human tumour cell types, and is involved in the induction of centrosome duplication-distribution abnormalities and aneuploidy in mammalian cells. STK15 amplification has been previously detected in breast tumour cell lines and in colon tumours; here, we report its amplification in approximately 12% of primary breast tumours, as well as in breast, ovarian, colon, prostate, neuroblastoma and cervical cancer cell lines. Additionally, high expression of STK15 mRNA was detected in tumour cell lines without evidence of gene amplification. Ectopic expression of STK15 in mouse NIH 3T3 cells led to the appearance of abnormal centrosome number (amplification) and transformation in vitro. Finally, overexpression of STK15 in near diploid human breast epithelial cells revealed similar centrosome abnormality, as well as induction of aneuploidy. These findings suggest that STK15 is a critical kinase-encoding gene, whose overexpression leads to centrosome amplification, chromosomal instability and transformation in mammalian cells.

High resolution analysis of DNA copy number variation using comparative genomic hybridization to microarrays.

Gene dosage variations occur in many diseases. In cancer, deletions and copy number increases contribute to alterations in the expression of tumour-suppressor genes and oncogenes, respectively. Developmental abnormalities, such as Down, Prader Willi, Angelman and Cri du Chat syndromes, result from gain or loss of one copy of a chromosome or chromosomal region. Thus, detection and mapping of copy number abnormalities provide an approach for associating aberrations with disease phenotype and for localizing critical genes. Comparative genomic hybridization (CGH) was developed for genome-wide analysis of DNA sequence copy number in a single experiment. In CGH, differentially labelled total genomic DNA from a 'test' and a 'reference' cell population are cohybridized to normal metaphase chromosomes, using blocking DNA to suppress signals from repetitive sequences. The resulting ratio of the fluorescence intensities at a location on the 'cytogenetic map', provided by the chromosomes, is approximately proportional to the ratio of the copy numbers of the corresponding DNA sequences in the test and reference genomes. CGH has been broadly applied to human and mouse malignancies. The use of metaphase chromosomes, however, limits detection of events involving small regions (of less than 20 Mb) of the genome, resolution of closely spaced aberrations and linking ratio changes to genomic/genetic markers. Therefore, more laborious locus-by-locus techniques have been required for higher resolution studies. Hybridization to an array of mapped sequences instead of metaphase chromosomes could overcome the limitations of conventional CGH (ref. 6) if adequate performance could be achieved. Copy number would be related to the test/reference fluorescence ratio on the array targets, and genomic resolution could be determined by the map distance between the targets, or by the length of the cloned DNA segments. We describe here our implementation of array CGH. We demonstrate its ability to measure copy number with high precision in the human genome, and to analyse clinical specimens by obtaining new information on chromosome 20 aberrations in breast cancer.

Genome scanning with array CGH delineates regional alterations in mouse islet carcinomas.

Carcinomas that develop in the pancreatic islets of transgenic mice expressing the SV40 T-antigens (Tag) under transcriptional control of the rat insulin II promoter (RIP) progress through well-characterized stages that are similar to aspects of human tumor progression, including hyperplastic growth, increased angiogenesis and reduced apoptosis. The latter two stages have been associated with recurrent loss of heterozygosity (LOH) and reduced genome copy number on chromosomes 9 (LOH9) and 16 (LOH16), aberrations which we believe contribute to these phenotypes. Earlier analyses localized LOH9 to approximately 3 Mb and LOH16 to approximately 30 Mb (both syntenic with human 3q21-q25) but were limited by low throughput and a lack of informative polymorphic markers. Here we show that comparative genomic hybridization to DNA microarrays (array CGH) overcomes these limitations by allowing efficient, genome-wide analyses of relative genome copy number. The CGH arrays used in these experiments carried BACs distributed at 2-20-MB intervals across the mouse genome and at higher density in regions of interest. Using array CGH, we further narrowed the loci for LOH9 and LOH16 and defined new or previously unappreciated recurrent regions of copy-number decrease on chromosomes 6, 8 and 14 (syntenic with human chromosomes 12p11-p13, 16q24.3 and 13q11-q32, respectively) and regions of copy-number increase on chromosomes 2 and 4 (syntenic to human chromosomes 20q13.2 and 1p32-p36, respectively). Our analyses of human genome sequences syntenic to these regions suggest that CYP24, PFDN4, STMN1, CDKN1B, PPP2R3 and FSTL1 are candidate oncogenes or tumor-suppressor genes. We also show that irradiation and genetic background influence the spectrum of aberrations present in these tumors.

Assembly of microarrays for genome-wide measurement of DNA copy number.

We have assembled arrays of approximately 2,400 BAC clones for measurement of DNA copy number across the human genome. The arrays provide precise measurement (s.d. of log2 ratios=0.05-0.10) in cell lines and clinical material, so that we can reliably detect and quantify high-level amplifications and single-copy alterations in diploid, polyploid and heterogeneous backgrounds.

Feasibility of using microbiology diagnostic tests of moderate or high complexity at the point - of - care in a delivery suite.

Point-of-care testing (POCT) is one of the fastest growing sectors of laboratory diagnostics. Most tests in routine use are haematology or biochemistry tests that are of low complexity. Microbiology POCT has been constrained by a lack of tests that are both accurate and of low complexity. We describe our experience of the practical issues around using more complex POCT for detection of Group B streptococci (GBS) in swabs from labouring women. We evaluated two tests for their feasibility in POCT: an optical immune assay (Biostar OIA Strep B, Inverness Medical, Princetown, NJ) and a PCR (IDI-Strep B, Cepheid, Sunnyvale, CA), which have been categorised as being of moderate and high complexity, respectively. A total of 12 unqualified midwifery assistants (MA) were trained to undertake testing on the delivery suite. A systematic approach to the introduction and management of POC testing was used. Modelling showed that the probability of test results being available within a clinically useful timescale was high. However, in the clinical setting, we found it impossible to maintain reliable availability of trained testers. Implementation of more complex POC testing is technically feasible, but it is expensive, and may be difficult to achieve in a busy delivery suite.

Neutrophil activation monitored by flow cytometry: stimulation by phorbol diester is an all-or-none event.

The population dynamics of single-cell stimulation was analyzed by monitoring autofluorescence by flow cytometry. Stimulation of the respiratory burst in human neutrophils by 12-O-tetradecanoyl phorbol-13-acetate (TPA) caused a decline in highly fluorescent cells (characteristic of resting neutrophils) and a corresponding increase in the number of weakly fluorescent cells (characteristic of activated neutrophils). Increasing concentrations of TPA caused increasing numbers of cells to shift from the highly fluorescent population to the weakly fluorescent population without the appearance of intermediate populations. Thus the neutrophil respiratory burst, a component of neutrophil cytotoxic response, is triggered in an all-or none fashion.

Comparative genomic hybridization for molecular cytogenetic analysis of solid tumors.

Comparative genomic hybridization produces a map of DNA sequence copy number as a function of chromosomal location throughout the entire genome. Differentially labeled test DNA and normal reference DNA are hybridized simultaneously to normal chromosome spreads. The hybridization is detected with two different fluorochromes. Regions of gain or loss of DNA sequences, such as deletions, duplications, or amplifications, are seen as changes in the ratio of the intensities of the two fluorochromes along the target chromosomes. Analysis of tumor cell lines and primary bladder tumors identified 16 different regions of amplification, many in loci not previously known to be amplified.

High-speed chromosome sorting.

Dual-beam high-speed sorting has been developed to facilitate purification of chromosomes based on DNA staining with the fluorescent dyes Hoechst 33258 and chromomycin A3. Approximately 200 chromosomes per second of two types can be sorted from a suspension of chromosomes isolated from human lymphoblasts while fluorescent objects (chromosomes, debris fragments, chromosome clumps, and nuclei) are processed at the rate of about 20,000 per second. This sorting rate is approximately ten times that possible with conventional sorters. Chromosomes of a single type can be sorted with a purity of about 90 percent. DNA from the sorted chromosomes is suitable for construction of recombinant DNA libraries and for gene mapping.

MRSA in children presenting to hospitals in Birmingham, UK.

Meticillin-resistant Staphylococcus aureus (MRSA) isolates from children presenting to Birmingham hospitals were characterized using molecular methods. The study was performed on MRSA isolates from children aged

Effects of methotrexate on transfected DNA stability in mammalian cells.

The chromosomal locations, amounts, and level of expression of transfected, amplified c-myc and dihydrofolate reductase sequences were measured in cells cultured in the presence and absence of methotrexate. These studies show that the location and amount of transfected sequences, as well as the level of expression, were more variable when the cells were cultured in methotrexate.

A new mouse tumor model system (RIF-1) for comparison of end-point studies.

A new tumor model system (RIF-1) was developed that is very suitable for studies in which clonogenic survival is compared with growth delay and control probability following various forms of treatment. The tumor was a radiation-induced sarcoma in the inbred female C3H/Km mouse. It had a low median tumor dose, had a satisfactory plating efficiency direct from in vivo to in vitro, was nonimmunogenic or minimally immunogenic, and metastasized only at a relatively advanced stage of growth. The cell line grew either as a monolayer on plastic dishes, as tumor spheroids in spinner culture, as lung nodules following injection of a single-cell suspension into the tail veins of syngeneic mice, or as a solid tumor. Both diploid and tetraploid clonogenic cells were found in monolayer cultures of the RIF-1 line.

Regulation of S49 lymphoma cell growth by cyclic adenosine 3':5'-monophosphate.

S49 lymphoma tissue culture cells arrest in the G1 phase of the cell cycle when treated with agents that elevate endogenous cyclic adenosine 3':5'-monophosphate (cAMP), such as cholera toxin or exogenously added active congeners of cAMP such as N6,O2'-dibutyryl cyclic adenosine 3':5'-monophosphate (Bt2cAMP). This phenomenon requires that cells contain the appropriate receptors: Mutant cells deficient in adenylyl cyclase fail to arrest in response to cholera toxin, and another mutant that lacks cAMP-dependent protein kinase does not respond to cholera toxin or to Bt2cAMP. The size distribution of cell populations treated with Bt2cAMP changes in a manner that reflects only the perturbation of cell cycle distribution. Arrested G1 cells in particular have the same volume as the G1 cells of an exponentially growing population. When G1 cells that have been arrested by Bt2cAMP are grown in fresh medium free of Bt2cAMP, they begin to reenter S phase after a delay of about 6 hr and do so with pseudo-first-order kinetics, with a half-life of 5 hr. These and other properties previously described suggest that cAMP regulates S49 cell growth by physiologically significant rather than artifactual mechanisms.

Cycle progression and division of viable and nonviable Chinese hamster ovary cells following acute hyperthermia and their relationship to thermal tolerance decay.

Cell cycle progression delays and subsequent growth kinetics of viable and nonviable Chinese hamster cells following acute (45.5 degrees) hyperthermia were documented in an attempt to correlate these changes with the decay of thermal tolerance. Following heating for various lengths of time, cells exhibited a delay in subsequent division which was related to cell survival by a power function relationship. A cell was considered to be viable if it retained the ability to divide to form a colony of 50 or more cells. The components of the delay in cycle transit for viable cells heated in G1 for a treatment of 20 min at 45.5 degrees were approximately 28 hr in G1 and 20 hr in S and G2-M. This represents a 7-fold decrease in the rate progression through G1 and a 2-fold decrease through S and G2-M relative to control rates. The doubling times of viable cells, in subsequent generations, were significantly decreased to a rate 61% of that of control up to 120 hr after heating. This reduction was in part due to lethal sectoring, i.e., a division which produces only one daughter that is capable of forming a viable subclone, within the viable progeny. Within a viable subclone, up to 30% of the cells that divided from 48 to 91 hr after a heat treatment of 20 min at 45.5 degrees were found to be nonclonogenic. Following resumption of division, nonviable cells slowly lost their capacity for proliferation. Nearly all thermal tolerance development induced by a 20-min pretreatment occurred while the viable cells remained in G1. Subsequent progression into heat-sensitive S and G2-M phases modulated thermal tolerance only slightly. Finally, maximal loss of thermal tolerance was exhibited at the time corresponding to the resumption of viable cell division.

Quantitative analysis of the cytokinetic response of KHT tumors in vivo to 1-beta-D-arabinofuranosylcytosine.

Quantitative estimates of cytokinetic perturbations induced over a 30-hr interval in KHT tumor cells in vivo by a single dose of 1-beta-D-arabinofuranosylcytosine (ara-C) were inferred from measurements of DNA distribution sequences, tritiated thymidine incorporation into tumor cell DNA, and radioactivity of cells labeled with tritiated thymidine prior to treatment with ara-C. These data were analyzed collectively to produce a mathematical model describing the effects of ara-C on tumor cell cycle kinetics. During analysis, we postulated that ara-C produced a transient block at the a G1-S-phase boundary, that ara-C killed all cells in S phase, and that the cells killed by ara-C did not cycle after treatment. The analysis showed that the duration of the G1-S-boundary block was 5.5 hr, that cells were recruited from G0 into cycle beginning 5.5 hr posttreatment, and that the G1, S, and G2M phase durations of the clonogenic cells which were initially 2, 11.5, and 2 hr, respectively, changed to 0.8, 4.6, and 0.8 hr at 14 hr posttreatment. Cells killed by ara-C were removed from the tumor beginning 10 hr posttreatment. We then used the mathematical model to predict clonogenic tumor cell survival following a second dose of ara-C administered at time intervals ranging from 1 to 30 hr after the first treatment. Clonogenic tumor cell survival following the second dose of ara-C was then experimentally determined and agreed well with the model predictions.

Decreased cytotoxicity of 1-beta-D-arabinofuranosylcytosine in 9L rat brain tumor cells pretreated with alpha-difluoromethylornithine in vitro.

The effect of pretreating 9L rat brain tumor cells in vitro with 10 mM alpha-difluoromethylornithine (DFMO), an enzyme-activated, irreversible inhibitor of ornithine decarboxylase, on the cytotoxicity of 1-beta-D-arabinofuranosylcytosine (ara-C) was investigated using a colony-forming assay. DFMO-mediated polyamine depletion significantly decreased the cytotoxicity of ara-C. The addition of putrescine to DFMO-pretreated cells replenishes intracellular polyamine levels and prevents the decrease in ara-C cytotoxicity. Flow cytometric analysis showed that cells treated with ara-C alone entered S phase semisynchronously within 8 hr after the end of treatment. This phenomenon was virtually eliminated by DFMO pretreatment. Addition of putrescine to DFMO-pretreated cells before treatment with ara-C prevented this DFMO-induced cell cycle kinetic effect.

Fluorescence-activated cell sorting analysis of the induction and expression of acute thermal tolerance within the cell cycle.

We have examined the cell cycle specificity of 45.5 degrees heat-induced toxicity and the induction and expression of thermal tolerance. Ultrapure populations of G1-, S-, and G2-M-phase cells were obtained through sequential centrifugal elutriation and flow cytometric cell sorting of Hoechst 33342-stained cells. We found no interaction of Hoechst 33342 with hyperthermia under staining conditions that gave good cytometric resolution of DNA distributions. Single dose-response survival curves indicated that S phase was the most sensitive to 45.5 degrees hyperthermia (Do = 1.97, 1.26, and 1.95 min for G1, S, and G2-M, respectively). Both S and G2-M phases exhibited a decreased ability from G1 to accumulate sublethal heat lesions as evidenced by decreased heat survival curve shoulders (Dq) = 13.7, 9.51, and 8.39 min for G1, S, and G2-M, respectively). Thermal tolerance, as measured by the decreased inactivation slope of the split-dose treatment, could be induced and expressed in G1, S, and G2-M phases. However, both the magnitude and temporal expression of tolerance were dependent on the position of the cell within the cell cycle at the time of the initial heat treatment. S-phase cells exhibited slightly less thermal tolerance as compared to G1 cells given isosurvival thermal induction doses as measured by the split-dose inactivation rate constants (heated/control = 8.37 and 5.62 for G1 cells at 12 and 24 hr and 7.68 and 5.27 for S-phase cells at 12 and 28 hr). Also, split-dose survival curves for cells heated in G2-M indicated a near total inability to accumulate heat-induced sublethal damage. Simultaneous bivariate (90 degrees light scatter and DNA content) progression analysis of heated replicates indicated that tolerance could probably be expressed in those cells which moved into other cycle compartments following the initial heat treatment. For instance, G1-phase cells preheated for 20 min began progression into normally heat-sensitive S phase between 24 and 28 hr after the heat treatment. This corresponded to approximately the time of maximal thermal tolerance expression. [3H]Thymidine suicide experiments also indicated that the ultimately clonogenic cells began movement into S phase at or near the time of maximal tolerance. In this case then, tolerance expression appeared to supersede the S-phase acute heat sensitivity. Heated S-phase cells began progression into G2-M between 4 and 12 hr, which corresponded temporally to large amounts of tolerance expression4 +

Measurement of sister chromatid exchanges at very low bromodeoxyuridine substitution levels using a monoclonal antibody in Chinese hamster ovary cells.

A monoclonal antibody to bromodeoxyuridine (BrdUrd) incorporated into DNA allowed visualization of sister chromatid exchanges (SCE) when as little as 0.6% of the thymine in a single DNA strand has been substituted. Measurement of the SCE frequency as a function of BrdUrd substitution in a normal Chinese hamster ovary cell line showed a plateau of six SCEs per cell for substitution levels up to at least 20%. A clear elevation in frequencies was noted at 60% substitution. However, in the mutant line EM9, previously shown to have a highly elevated frequency of SCE, the level of exchanges declined continuously as the percentage of BrdUrd substitution decreased. At 0.6% substitution, the frequency of SCE was still 4-fold higher than that of the parental cells. The antibody procedure described here should be useful in evaluating the extent to which SCEs induced by mutagenic agents result from interactions between the DNA damage caused by the agent and the BrdUrd routinely used for measuring SCE.

Effect of cell cycle position on the survival of 9L cells treated with nitrosoureas that alkylate, cross-link, and carbamoylate.

The relationship between cell cycle position and cytotoxicity was studied in 9L rat brain tumor cells treated with nitrosoureas that, depending on their structures, can alkylate or alkylate and cross-link DNA and/or carbamoylate biomolecules. Because pure populations of G1-, S-, and G2-M-phase cells could not be obtained with the centrifugal elutriation methods used, drug sensitivity of cells in each phase of the cell cycle was estimated using a mathematical model that accounts for variation in enrichment of elutriated fractions. 1,3-Bis(2-chloroethyl)-1-nitrosourea, 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea, 1-(2-chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitrosourea, 1-(2-chloroethyl)-3-(2,6-dioxo-3-piperidyl)-1-nitrosourea, which alkylate and cross-link DNA and carbamoylate biomolecules, and 2-[3-(2-chloroethyl)-3-nitrosoureido]-D-glucopyranose (chlorozotocin), which alkylates and cross-links DNA but cannot carbamoylate biomolecules, killed more cells in G1 and G2-M phases than in S phase. N-Ethylnitrosourea, which alkylates and carbamoylates but does not form DNA interstrand cross-links, was more toxic to cells in S phase than in other phases. Cell kill caused by N,N'-bis(trans-4-hydroxycyclohexyl)-N-nitrosourea, a compound that carbamoylates only, increased progressively through the cell cycle from G1 to M. Nitrosoureas that cross-link DNA were more cytotoxic than nitrosoureas that do not cross-link DNA, although the latter had phase specificity. The results suggest that the increased sensitivity of G1- and G2-M-phase cells to chloroethylnitrosoureas is related to the formation of DNA interstrand cross-links.