Tumour regression observed in many conditional mouse models following oncogene inactivation provides the impetus to develop, and a platform to preclinically evaluate, novel therapeutics to inactivate specific oncogenes. Inactivating single oncogenes, such as c-Myc, can reverse even advanced tumours. Intriguingly, transient c-Myc inactivation proved sufficient for sustained osteosarcoma regression; the resulting osteocyte differentiation potentially explaining loss of c-Myc's oncogenic properties. But would this apply to other tumours?L over-expression or a favourable microenvironment, respectively. Moreover, tumours progress despite reacquiring a differentiated phenotype and partial loss of vasculature during c-Myc inactivation. Interestingly, reactivating c-Myc in β-cell tumours appears to result not only in further growth of the tumour, but also re-expansion of the accompanying angiogenesis and more pronounced β-cell invasion (adenocarcinoma).We show that brief inactivation of c-Myc does not sustain tumour regression in two distinct tissue types; tumour cells in pancreatic islets and skin epidermis continue to avoid apoptosis after c-Myc reactivation, by virtue of Bcl-xGiven that transient c-Myc inactivation could under some circumstances produce sustained tumour regression, the possible application of this potentially less toxic strategy in treating other tumours has been suggested. We show that brief inactivation of c-Myc fails to sustain tumour regression in two distinct models of tumourigenesis: pancreatic islets and skin epidermis. These findings challenge the potential for cancer therapies aimed at transient oncogene inactivation, at least under those circumstances where tumour cell differentiation and alteration of epigenetic context fail to reinstate apoptosis. Together, these results suggest that treatment schedules will need to be informed by knowledge of the molecular basis and environmental context of any given cancer. The ability to switch expression of a given oncogene 'on' or 'off' ewed in: ,2). Giveewed in: ). Similaewed in: ,5.Several studies using conditional mouse models of various cancers have unexpectedly shown that inactivation of the initiating oncogene is sufficient for reversal not only of the primary tumour but also of invasive and metastatic lesions, many of which contain multiple genetic and epigenetic alterations -18.The tumour regression observed in many of these models following sustained oncogene inactivation provides a powerful platform on which to build a deeper understanding of fundamental tumour biology and with which to preclinically evaluate novel therapeutics to target specific genes. A recent study has shown that brief inactivation 10 days) of c-Myc was sufficient for the sustained regression of c-Myc induced invasive osteogenic sarcomas in transgenic mice ; subsequ0 days ofL overexpression) induced β-cell growth arrest and re-differentiation into mature β-cells, accompanied by the collapse of tumour vasculature and tumour cell mass resulting from apoptosis, despite the constitutive expression of Bcl-xL in the tumour cells [Previously, we have shown that sustained c-Myc inactivation in locally invasive pancreatic islet tumours (induced by c-Myc activation in β-cells on a background of Bcl-xur cells . Similarur cells . HoweverL) as is the case in the islet tumourigenesis model, or by the presence of survival cues in the microenvironment, as seen in c-Myc-induced skin tumours. Second, we already know that sustained c-Myc inactivation leads to regression in both cases.These conditional models, in which c-Myc-induced tumours of pancreatic islets and skin epidermis can be initiated at any given time in the adult animal, were chosen for the studies presented here and offer several advantages (reviewed in ). First,in vivo. In contrast to the osteogenic sarcoma model, re-activating c-Myc in islet tumours does not lead to accelerated β-cell apoptosis, but rather restores the oncogenic properties of c-Myc, rapidly re-initiating β-cell proliferation, loss of differentiation, loss of E-cadherin, local invasion and angiogenesis. This occurs despite the re-differentiation of previously c-Myc-activated tumour cells to a more mature phenotype and the loss of some of the newly acquired vasculature, occurring during the period of c-Myc inactivation. Moreover, as no new β-cells arise during the period of c-Myc inactivation, replication is probably restored in those same cells that have previously experienced c-Myc activation. Similarly, in epidermis, reactivating c-Myc in suprabasal keratinocytes does not result in apoptosis, which remains confined to the shedding areas of parakeratosis at the skin surface, but restores the papillomatous phenotype, inducing cell proliferation and dysplasia. These results are in line with a very recent study in a different system, published while this manuscript was under consideration. Shachaf and colleagues demonstrated that invasive c-Myc-induced hepatocellular carcinomas regress when c-Myc expression is turned off but, interestingly, some tumour cells remain 'dormant' even for prolonged periods and contribute to cancer progression if c-Myc expression is subsequently reinitiated [Here we show the consequences of inactivating c-Myc transiently, for a period of 4 to 9 days, in these distinct tumour types nitiated .Taken together, these findings suggest that a cautious approach is required in considering cancer therapies aimed at transient oncogene inactivation. First, a more comprehensive understanding of the genetic basis and environmental context of any individual tumour would be required in order to predict the likely success of such a treatment schedule. Second, at least under those circumstances where tumour cell differentiation and alteration of epigenetic context would not be predicted to reinstate apoptosis and no alternative mechanism exists for tumour cell removal, sustained inactivation of the offending oncogene would seem the desired therapeutic goal.TAM (c-Myc) in β-cells of adult pIns-c-MycERTAM mice cross-bred with RIP7-Bcl-xL mice [Islet tumours were induced in nine mice by activation of c-MycER-xL mice  by daily-xL mice . Three mpIns-c-MycERTAM/RIP-Bcl-xL double transgenic mice exhibited normal islet morphology  led to restoration of the oncogenic properties of c-Myc . Sections were cut throughout the pancreas and selected sections at every 100 μm were stained with H&E for histological analysis as well as with immunohistochemical markers for proliferation (Ki-67), differentiation (insulin), apoptosis (TUNEL) and loss of cell-cell contact (E-cadherin). Importantly, pancreatic islets following 14 days of c-Myc reactivation showed no signs of tumour regression but rather progression of islet tumourigenesis, with the vast majority of islets showing more pronounced invasion (adenocarcinoma) Figure .TAM (c-Myc) in suprabasal keratinocytes of adult Involucrin-c-MycERTAM transgenic skin by daily topical administration of 4-OHT for 14 days as previously described [Development of papillomatosis  was induced in nine mice following activation of c-MycERescribed . Three mInvolucrin-c-MycERTAM transgenic mice exhibited normal skin morphology , then as long as c-Myc inactivation induces differentiation, and, presumably, no anti-apoptotic mutation has been acquired, a transient period may suffice for sustained tumour regression. However, in many cases where an anti-apoptotic lesion is also present , or potentially the microenvironment continues to prevent apoptosis, sustained inactivation would be essential for tumour regression. Moreover, it can also be stated that partial reversal of angiogenesis, at least in the islet tumours, will not have any lasting impact on tumour progression if angiogenesis automatically continues apace of further growth of the tumour. Finally, it seems likely that reacquisition of a differentiated phenotype does not preclude previously c-Myc activated cancer cells from re-exhibiting cancer behaviour once c-Myc is reactivated – removal of these cells by apoptosis or other means would seem necessary to remove the threat of cancer recrudescence.Identifying these key differences in behaviour between different cell types/developmental stages is not an academic exercise, but can give vital information about the mechanisms and context whereby oncogene activity may be determined, which in addition to the biological interest might also provide new knowledge of direct relevance to human cancer.Given the fact that deregulation of c-Myc expression is one of the most frequently described abnormalities in human cancers and has been observed in β-cell derived tumours and in human skin epidermal tumours -32, our In several transgenic mouse models of cancer, the generation of invasive/metastatic tumours in which more than one genetic event was involved can be reversed following inactivation of a single oncogene. These findings suggest that some metastatic lesions may remain responsive to therapeutic intervention originally targeted to the primary lesion. Recent data from a mouse model of osteogenic sarcoma showed that even transient c-Myc inactivation can result in sustained tumour regression .in vivo. In contrast to the osteogenic sarcoma model, re-activating c-Myc in islet β-cell tumours does not lead to accelerated β-cell apoptosis, but rather restores the oncogenic properties of c-Myc, rapidly re-initiating β-cell proliferation, loss of differentiation, loss of E-cadherin, local invasion and angiogenesis. This occurs despite the re-differentiation of previously c-Myc-activated tumour cells to a more mature phenotype and the loss of some of the newly acquired vasculature, occurring during the period of c-Myc inactivation. Similarly, in epidermis, reactivating c-Myc in suprabasal keratinocytes does not result in apoptosis, which remains confined to the shedding areas of parakeratosis at the skin surface, but restores the papillomatous phenotype, inducing cell proliferation and dysplasia.Here we show the consequences of inactivating c-Myc transiently in two distinct tumour types The differences between the conditional tumour models used by ourselves and Jain et al. , rather Together, these results suggest that treatment schedules will need to be informed by knowledge of the molecular basis and environmental context of any given cancer.pIns-c-MycERTAM and Involucrin-c-MycERTAM mice were generated by cloning a full-length human c-myc cDNA fused to the hormone-binding domain of a modified estrogen receptor (c-MycERTAM) downstream of the rat insulin promoter and the human involucrin promoter, respectively, as previously described [escribed ,9. DNA cRIP7-Bcl-xL mice were obtained from Dr Doug Hanahan [c-MycERTAM cDNA: (forward) 5' CCA AAG GTT GGC AGC CCT CAT GTC 3'; (reverse) 5' AGG GTC AAG TTG GAC AGT GTC AGA GTC 3'. PCR program: 94°C 2 min 1 cycle, [94°C 1 min, 57°C 1 min, 72°C 2 min] 30 cycles, 72°C 10 min 1 cycle. PCR product size: 413 bp. Primers used for the detection of RIP7-Bcl-xL cDNA: (forward) 5' AGC ACT TTC TGC AGA CCT AGC AC 3'; (reverse) 5' CAG CTC CCG GTT GCT CTG AGA C 3'. PCR program: [94°C 1 min, 60°C 30 s, 72°C 2 min] 30 cycles, 72°C 3 min 1 cycle.Heterozygous  Hanahan . Littersad libitum.Transgenic mice were housed under barrier conditions with a 12 hour light/dark cycle and access to food and water TAM, was targeted to pancreatic β-cells using a rat insulin promoter, or to suprabasal keratinocytes using the human involucrin promoter. As shown in our previous publications [TAM protein remains inactive due to association of the cells' own hsp90 with the ERTAM. Upon administration of 4-hydroxytamoxifen (4-OHT), hsp90 is displaced allowing association of c-Myc's partner, Max, to form transcriptionally active heterodimers [Expression of the chimeric protein, c-MycERications ,9, the trodimers .TAM protein in pancreatic β-cells of adult transgenic mice, 1 mg of 4-OHT (Sigma) sonicated in peanut oil (1 mg/0.2 ml) was administered daily by IP injection. To activate c-MycERTAM protein in skin epidermis of adult transgenic mice, 1 mg of 4-OHT (Sigma) dissolved in ethanol (1 mg/0.2 ml) was administered daily by topical application to a shaved area of dorsal skin.To activate c-MycERTAM protein was achieved following withdrawal of 4OHT. As c-MycERTAM RNA and protein levels remain unchanged in the presence or absence of 4OHT, Northern and Western blot analysis will not confirm whether the protein is inactive. Thus, to confirm inactivity of the c-MycERTAM protein in pancreatic β-cells, we show reversal of several markers of Myc activation by day 4 of 4OHT withdrawal -growth arrest, re-differentiation, re-establishment of cell-cell contact – by immunohistochemistry (see ref [Inactivation of c-MycER(see ref  and ResuTAM protein in skin epidermis was confirmed using immunohistochemistry for markers of re-differentiation (K1 and K14) and growth arrest (see Results section). The tight regulation of the c-MycERTAM protein in skin epidermis was also previously shown in [in situ hybridisation for detection of ODC RNA, a known c-Myc target gene; by day 5 following withdrawal of 4OHT, ODC RNA is no longer detected.Similarly, inactivation of c-MycERshown in  using inPancreata or skin were excised from mice and 5–10 mm pieces of tissue were fixed overnight in neutral-buffered formalin, embedded in paraffin wax and sectioned (5–10 μm). Frozen sections were prepared from tissue embedded in OCT and frozen in foil on a bath of dry ice and ethanol. Prior to staining, frozen sections were air-dried and fixed in 4% paraformaldehyde for 15 minutes. Alternatively, for frozen sections, tissue was fixed in 4% paraformaldehyde for 2 hours followed by incubation in 30% sucrose overnight at 4°C. For pancreata analysis, sections (5–10μm) were cut throughout the entire pancreas and every tenth section was selected for histological and immunohistochemical examination. For skin, sections (5–10μm) were cut through two 10 mm pieces of tissue and every tenth section was selected for analysis.Primary antibodies were as follows: rabbit polyclonals Ki-67 (Novacastra) and Nkx6.1 (Ole Madsen, NovoNordisk); rabbit anti-mouse laminin (Sigma); guinea-pig anti-porcine insulin (Dako); rat anti-mouse E-cadherin, (Zymed); rabbit anti-mouse keratin 1 (BabCo); rat anti-mouse CD45 (AbCam). E-cadherin and laminin antibodies were found to label reliably only frozen tissue sections. Other antibodies were effective when used on both paraffin-embedded and frozen sections, although Ki-67 and Nkx6.1 required epitope retrieval by microwaving paraffin-embedded sections at 700 W for 2 × 10 minutes in 0.01 M citrate buffer, pH6.0 (Vector) followed by immersion in cold water.Antibodies were diluted in incubation buffer: PBS/0.5% Triton X-100 containing 1:25 dilution of serum from the same species as the secondary antibody. Primary antibodies for insulin and Ki-67 were applied together to sections for 1 hour. Sections were then incubated in Texas Red-conjugated goat anti-guinea pig Ig secondary antibody together with FITC-conjugated goat anti-rabbit secondary antibody (Vector). After washing, sections were mounted in Vectashield mounting medium (Vector).To detect cells undergoing apoptosis, costaining with TUNEL/insulin, TUNEL/laminin and TUNEL/K1, immunofluorescent staining was performed by applying insulin, laminin or K1 antibodies to sections for 1 hour at room temperature followed by Texas Red-conjugated goat anti-guinea pig (for insulin antibodies) and goat anti-rabbit (for laminin and K1 antibodies) Ig secondary antibody (Vector). TUNEL staining was subsequently performed using ApopTag Fluorescein Direct kit (Chemicon) for frozen tissue sections and ApopTag Fluorescein Indirect kit (Chemicon) for paraffin-embedded tissue sections.SP participated in the design of the study, administered 4OHT to relevant mice, collected tissue, carried out immunohistochemical staining, coordinated and analysed data, drafted the manuscript, and provided part of the funds. SA carried out genotyping, assisted with the administering of 4OHT to mice, collected tissues, cut sections and assisted with capturing of images. LC assisted with genotyping, cutting of sections, and immunohistochemical staining. VI assisted with genotyping and immunohistochemical staining. SZ assisted with genotyping and immunohistochemical staining. MK participated in the design of the study, assisted with the coordination and analyses of data, helped draft the manuscript and provided funding.


Chronic meningitis is defined as symptoms and signs of meningeal inflammation and persisting cerebrospinal fluid abnormalities such as elevated protein level and pleocytosis for at least one month.Neisseria meningitidis group B. The patient made a dramatic recovery with high-dose intravenous ceftriaxone antibiotic therapy for meningococcal meningitis.A 62-year-old woman, of unremarkable past medical history, was admitted to hospital for investigation of a four-week history of vomiting, malaise an associated hyponatraemia. She had a low-grade pyrexia with normal inflammatory markers. A CT brain was unremarkable and a contrast MRI brain revealed sub-acute infarction of the right frontal cortex but with no evidence of meningeal enhancement. Due to increasing confusion and patient clinical deterioration a lumbar puncture was performed at 17 days post admission. This revealed gram-negative coccobacilli in the CSF, which was identified as 1) Chronic bacterial meningitis may present highly atypically, particularly in the older adult. 2) There may be an absent or reduced febrile response, without a rise in inflammatory markers, despite a very unwell patient. 3) Early lumbar puncture is to be encouraged as it is essential to confirm the diagnosis.4) Despite a delayed diagnosis appropriate antibiotic therapy can still lead to a good outcome. Bacterial Meningitis usually presents as an acute illness, predominantly affecting children and young adults. It typically presents with the classical clinical triad of fever, neck stiffness and an altered mental state. However, it may rarely present as a chronic illness, without the classic clinical features noted in acute meningitis.A 62-year-old retired woman was admitted to hospital via her GP for investigation of a four-week history of vomiting and malaise associated with hyponatraemia. She was initially diagnosed as suffering from viral gastroenteritis. However, the vomiting had persisted and had become associated with a mild frontal headache. She had an unremarkable past medical history and was not taking any regular medication. She had never smoked and there was no recent antecedent foreign travel.On examination she appeared clinically dehydrated but otherwise looked well, and was alert and orientated. She was apyrexial and had no rash, photophobia, neck stiffness or stigmata of endocarditis. She had a sinus tachycardia of 104/minute, with normal heart sounds, and a blood pressure of 130/76 mmHg. Chest, abdominal and neurological examinations were unremarkable. She had a plasma sodium of 127 mmol/L (135–145 mmol/L), potassium of 3.4 mmol/L (3.5–5.0 mmol/l), urea 4.8 mmol/L (3.0–6.5 mmol/L) and creatinine of 68 mmol/L (60–125 mmol/L). There was no biochemical evidence of the syndrome of inappropriate antidiuretic hormone (SIADH) production . Serum complement and plasma immunoglobulin levels were unremarkable with no evidence of immunosuppression. In addition, she had a normal full autoimmune profile and thyroid function. Random cortisol level was mildly elevated at 799 nmol/L  consistent with a stress response.9/L  with a neutrophilia of 10 × 109/L . Her ECG and chest X-ray were normal. Her C-reactive protein (CRP) was slightly elevated at 10 mg/L  and erythrocyte sedimentation rate (ESR) was normal at 5 mm/hour. Her Chest X-ray and electrocardiogram were normal. Initial microbiological investigations (blood cultures, urine microscopy and culture) were normal.Her initial white cell count (WCC) was mildly elevated at 13.0 × 10Initial management consisted of slow intravenous rehydration with normal saline and antiemetic therapy, which led to a mild symptomatic improvement. Upper gastrointestinal endoscopy revealed mild oesophagitis.9/L). However, on day 4 of admission she developed a low-grade pyrexia of 37.5°C, which persisted (<38°C). A CT scan of the head revealed periventricular patchy white matter changes but no features of raised intracranial pressure or space occupying lesion.During the ensuing two weeks her laboratory investigations remained stable . In addition, her nausea and vomiting had failed to fully settle with supportive treatment. The LP results were as follows: cerebrospinal fluid (CSF) appearance was pale yellow and clear; protein = 5.69 g/L (0.15–0.4 g/L); CSF glucose 1.7 mmol/L versus plasma glucose 5.7 mmol/L ; CSF WCC = 106/mL  – 99% lymphocytes. Gram's stain revealed gram-negative coccobacilli; acid-fast bacilli were not seen. She was commenced on intravenous ceftriaxone.On day 18 she underwent a lumbar puncture (LP) as she still had a low-grade pyrexia (temperature 37.5°C) and neutrophilia of 9.3 × 10Contrast MRI brain revealed sub-acute infarction of the right frontal cortex but with no evidence of meningeal enhancement. EEG demonstrated slow wave activity, which was consistent with a meningo-encephalitis.Neisseria meningitidis group B, type NT, subtype NT P1.16/nt. She underwent contact tracing and completed a 10-day course of intravenous ceftriaxone. She continued to make a slow but progressive recovery. After a period of rehabilitation and intense physiotherapy she was discharged home 40 days after admission, with mild residual gait ataxia.Within 48 hours of intravenous antibiotics she was more alert, orientated, and sitting out of bed. CSF culture grew gram-negative cocci, which was identified as This case report presents two important clinical concepts: firstly, the presentation of chronic meningitis and secondly, the clinical presentation of bacterial meningitis in the older adult (defined as > 60 years old). The diagnosis was delayed due to the highly atypical clinical presentation .Chronic meningitis is defined as symptoms and signs of meningeal inflammation and persisting cerebrospinal fluid (CSF) abnormalities such as elevated protein level and pleocytosis for at least one month ,3. It afThere are several distinguishing features that may help to differentiate chronic meningitis from adult acute bacterial meningitis table . The claHyponatraemia (as in our patient), whilst very uncommon in acute bacterial meningitis, is seen in the vast majority of cases of chronic meningitis ,11. AlthAcute bacterial meningitis is usually a rapidly progressive and highly lethal disease in older adults . Rapid dGiven the success of childhood immunization, and an increasingly aging population, the proportion of older adults presenting with bacterial meningitis is increasing . There aThe clinical presentation of bacterial meningitis is more variable in the older as compared with the younger adult, with fewer patients manifesting with the classic symptoms of fever, neck stiffness and altered mental state than among younger adults . It has Listeria monocytogenes meningitis or alternative causes such as tuberculous and fungal infection [Our patient's CSF showed a lymphocytosis, raised protein, and low glucose ratio, which are seen in only 10% of bacterial meningitis cases. This CSF profile would normally suggest infection with nfection ,31.Neisseria (N) meningitidis is a leading cause of bacterial meningitis in the Western World and tends to predominate in young adults [N. meningitidis is a gram-negative, aerobic diplococcus. It is classified into serogroups (e.g. A,B,C etc) according to the immunological reactivity of their polysaccharides [g adults ,20,25. Ncharides ,20. The charides . Meningocharides -37.This case highlights the diagnostic challenge associated with bacterial meningitis presenting in an older patient. The presentation was made even more difficult owing to the blunted febrile response, the lack of inflammatory response observed in laboratory tests and the chronicity of the patient's symptoms. The diagnosis required thorough investigation during the inpatient stay. Early lumbar puncture is to be encouraged as it is essential to confirm the diagnosis. Despite a delayed diagnosis appropriate antibiotic therapy can still lead to a good outcome.The authors declare that they have no competing interests.MD generated the idea of writing the case report and was the consultant in charge of the patient. CD reviewed the case notes of the patient and wrote the original draft of the case presentation. CB significantly revised the original draft and added the conclusions, references and figures. AH offered considerable help with the manuscript revisions. All authors contributed to the final version of the manuscript.The pre-publication history for this paper can be accessed here:


Inter-individual variation in normal human mammary epithelial cells in response to oxythioquinox (OTQ) is reported. Gene expression signatures resulting from chemical exposures are generally created from analysis of exposures in rat, mouse or other genetically similar animal models, limiting information about inter-individual variations. This study focused on the effect of inter-individual variation in gene expression signatures.p value ≤ 0.05 in three of four cell strains analyzed.Gene expression was studied in primary normal human mammary epithelial cells (NHMECs) derived from four women undergoing reduction mammoplasty [Cooperative Human Tissue Network ]. Gene transcription in each cell strain was analyzed using high-density oligonucleotide DNA microarrays (HuGeneFL, Affymetrix™) and changes in the expression of selected genes were verified by real-time polymerase chain reaction at extended time points (ABI). DNA microarrays were hybridized to materials prepared from total RNA that was collected after OTQ treatment for 15, 60 and 120 min. RNA was harvested from the vehicle control (DMSO) at 120 min. The gene expression profile included all genes altered by at least a signal log ratio (SLR) of ± 0.6 and p53 polymorphisms. The two strains expressing the major variant of p53 had 83 common genes altered (35 increased, 48 decreased) at one or more time point by at least a 0.6 signal log ratio (SLR). The intermediate variant strains showed 105 common genes altered (80 increased, 25 decreased) in both strains.RNA species were clustered in various patterns of expression highlighting genes with altered expression in one or more of the cell strains, including metabolic enzymes and transcription factors. Of the clustered RNA species, only 36 were found to be altered at one time point in three or more of the cell strains analyzed (13 up-regulated, 23 down-regulated). Cluster analysis examined the effects of OTQ on the cells with specific Differential changes in expression of these genes may yield biomarkers that provide insight into inter-individual variation in cancer risk. Further, specific individual patterns of gene expression may help to determine more susceptible populations. Oxythioquinox (Morestan™ or OTQ, Bayer Corp) is a prototypical pesticide that was first used in 1968 on crops such as apples, pears, cucumbers, and gherkins. However, its use was later confined to non-food crops, limiting exposure to nursery and greenhouse employees. OTQ is a member of the quinoxaline class of pesticides, which also includes chlorquinox and thioquinox. Principal agricultural use of OTQ was limited to the states of California, Washington, Florida, New York, Pennsylvania, Ohio and Michigan ,3. Furthin vivo studies in rats found alterations of a variety of metabolic enzymes following OTQ exposures, including alkaline phosphatase [Scientifically acceptable toxicity studies of OTQ are sparse. Early sphatase . Althougsphatase  suggest sphatase  showed tsphatase . OTQ wassphatase . OTQ hassphatase . Howeversphatase ,11-13. Ab]pyridine (PhIP) and 7,12-dimethylbenz [a]anthacene (DMBA), and was able to show that while both chemicals altered expression in some genes in a similar fashion, each induced unique gene expression patterns.This study is similar to work currently being carried out to determine gene expression profiles of a variety of environmental agents, including chemicals, physical agents and physiologic stresses -19. The The primary goal of this study was to look for consistent changes common to all donors that could potentially be used as biomarkers of exposure, creating a gene expression profile following OTQ exposure in normal human cells. Given previous research in hepatoxicity, ideally normal human liver cells would have been used as a model system. However, we needed a normal human tissue that was readily accessible. Therefore, gene expression was studied in primary normal human mammary epithelial cells from four different donors in response to OTQ exposure. Current microarray analysis of pesticide exposure focuses on animal studies, not allowing for analysis of inter-individual variation. With the use of microarrays including clinical diagnosis, genetic susceptibility to disease and treatment, the need to determine the potential role of inter-individual variation on gene expression patterns in all potentially exposed tissues. Genes found to be altered in multiple cell strains could be considered biomarkers for individual populations. Given the small number of cell strains used, follow-up analysis in a larger number of samples is required to confirm any potential biomarkers. Biomarkers derived from this study could potentially be used in future epidemiology studies analyzing the effects of pesticide exposure. Further, this gene expression profile could be compared to those of other pesticides and of known carcinogens to develop a more detailed mechanistic definition of these chemicals.2.Primary normal human mammary epithelial cells (NHMECs) were derived from tissues salvaged at reduction mammoplasty obtained though the Cooperative Human Tissue Network . Development and characterization of cell strains was achieved using standard methods . Cells wp53 expression with minimal toxicity at 2 hours with a final concentration of 6.25 μM. Cells were treated by diluting the stock OTQ/DMSO mixture in media and adding this solution to aspirated cells, allowing even exposure to all cells. DMSO (0.001%) alone was used as a vehicle control. At the end of the treatment period, cells were removed for RNA isolation. Cell viability was determined by Trypan Blue exclusion assay.Treatment was performed on cells in passage six at 70% confluency, as routinely performed in our laboratory. Preliminary studies analyzed a range of OTQ concentrations (0 – 12.5 μM) selected based on previous research ,7 and tip53 antibody (1:1000, DO-1, Santa Cruz Biotech, Santa Cruz, CA) and incubated overnight at 4°C. The next day, the media was again removed and the secondary antibody (1:1500, goat anti-mouse FITC, Santa Cruz Biotech) was incubated for one hour at room temperature. Slides were washed in triplicate with phosphate buffered saline (PBS) and cover slips were added. Slides were dried for one hour at room temperature before viewing, using the laser scanning confocal microscope BX50 (Olympus), and quantitative analysis was performed [p53 expression was quantified between different time points and strains by determination of the area under the integrated intensity curve (Fluoview, Olympus, B & B Microscopes, Pittsburgh, PA).Confluent cells in passage five were trypsinized and plated on eight-well slides (LabTek II Slide System, Nunc, Naperville, IL). These cells were grown to 70% confluency before being treated with OTQ (6.25 μM) for 15, 60 and 120 min or DMSO (0.001%) for 120 min. At the end of treatment, the media was aspirated and the cells were fixed with methanol. Slides were then stained with anti-erformed . RelativMicroarray analysis was performed in duplicate using the HuGeneFL high-density oligonucleotide microarrays (Affymetrix™, Santa Clara, CA). Protocols from the Affymetrix Expression Analysis Technical Manual were followedin vitro transcription (IVT) reaction  was then performed to produce biotin-labelled cRNA from the cDNA. Excess biotinylated dUTPs were removed by RNEasy Mini Kit before being fragmented and added to a hybridization cocktail, including Eukaryotic Hybridization controls (Affymetrix), BSA and herring sperm DNA (Gibco, Grand Island, NY) and biotinylated anti-streptavidin antibody (Vector Laboratories, Burlingame, CA). Hybridization on microarrays was performed for 16 hours at 45°C in the Gene Chip Hybridization Oven with rocker (Affymetrix).RNA was isolated from cells with Trizol (Gibco, Grand Island, NY), followed by purification with RNEasy Mini Kit . Spectrophotometer measurements were required to give a 260/280 ratio of 1.9–2.1 for use in microarray analysis. Double-stranded cDNA was then synthesized from total RNA (Superscript Choice System, Invitrogen, Carlsbad, CA). An th position to control for non-specific binding (mismatch, MM). Results are given in signal intensities with a p-value determined from perfect match/mismatch (PM/MM) intensities by Tukey's Biweight analysis. Each array was normalized to a scaling factor of 1500 to correct for array variation. All arrays for each cell strain were analyzed on the same day to minimize variation.Microarrays were washed and stained using the protocol, as described in the Affymetrix Manual, with the GeneChip Fluidics Station 400 (Affymetrix). Arrays were then scanned with the Affymetrix Scanner . Expression profiles were analyzed using Microarray Suite 5.0, MicroDB 3.0 and Data Mining Tool 3.0 (Affymetrix). Affymetrix arrays are produced using multiple 25-mer oligonucleotides (11–20 per target gene). Each oligonucleotide is created to match the selected region of the target gene (perfect match, PM), while a similar oligonucleotide is created altered in the 13p value of 0.05 or less at one or more time points. Analysis was performed to comply with MIAME standards.Signal log ratio was determined by comparison of the signal intensities for the baseline (vehicle control) and the treatment array. This is computed using a one-step Tukey's Biweight method by taking a mean of the log ratios of probe pair intensities across the two arrays. This method helps to filter out differences due to different probe binding coefficients that may lead to false positives and/or negatives. A signal log ratio of zero represents no change in gene expression as a result of OTQ exposure. A signal log ratio of one is equivalent to a fold change of two between the treatment and control. The results described here are the average of both duplicates, with the average percent variability between duplicate arrays being 1.5% . Only relative changes equal to or greater than 0.6 signal log ratio (SLR) were considered a significant change as a result of exposure. The biological significance of each change is determined with Wilcoxon's signed rank test with the Affymetrix software. Gene chip analysis was performed by self-organizing map (SOM) clustering, focusing on genes with a detection ® (ABI) to yield unique fragments for each gene under study. Reactions were set up following recommended protocols using 100 pmol of each primer (Sigma-Genosys) and approximately 60 ng template per reaction. Reactions were performed in duplicate for each sample for 40 cycles . Fold change was determined based on average cycle threshold (CT) values for all duplicates and converted to signal log ratio.cDNA synthesized from each sample as in the Affymetrix analysis (Invitrogen) was used in a one-step RT-PCR analysis reaction. Analysis was performed in duplicate on the ABI 7700 cycler, with the SYBR Green Master Mix (ABI) and samples were normalized using both 18S and GAPDH expression levels for each sample. Primers were designed using Primer ExpressTrypan Blue was used to analyze toxicity by measuring cell viability for each cell strain for each treatment. The results showed a range of viability from 92–97% at all time points, except for the last time point in strain 3, which had a viability of only 65% at 120 min (results not shown). This decrease in viability at 120 min was not found to be directly correlated to p53 or p21 protein expression, or to any particular gene expression pattern. The dose of OTQ used was based on these findings.p53 protein levels were visually compared to those after treatment in each cell strain. Integrated fluorescence intensities were measured on each optical slice of cells. The fluorescence was determined as the area under the curve in arbitrary units (AU)  showed a 10-fold lower p53 expression at each time point.Baseline ) Figure . This rep53, despite the increase in p53 protein levels observed by immunofluorescence. Studies of benzo [a]pyrene exposure in our laboratory have also found similar results for p53 expression [DNA microarray analysis found no change in pression . The effAlthough inter-individual variation between donors in response to OTQ was evident, there were also some genes found to be increased consistently in all strains by microarray analysis Table . Self-or.SOM clustering was used to show patterns of expression in each cell strain, and followed by further subclustering of those clusters of interest across all cell strains Figure . This ancytochrome P4502A13 (CYP2A13) and dihydrodiol dehydrogenase. Although expression was slightly increased at one time point, the temporal patterns were slightly different , immune response (cyclophilin), and apoptosis (MAD-3). These genes showed a consistent increase in expression following exposure to OTQ (Table MAP kinase kinase), cell metabolism genes (S-adenosylmethionine synthetase, cytochrome bc-1), and transcription factors (E1A enhancer).From the full list of genes that fit these criteria, selected genes were chosen due to their potential role in carcinogenesis, whether by cell cycle control, immune response or other specific functions. Functions were described as annotated by NetAffx . These graf oncogene, GADD45, EWS, Cyclin D1) as well as immune response (immunophilin, gro-β), cell proliferation (TGFβ), and RNA processing (HnRNP F protein).Some genes of interest were variably altered by strain, examples of which are also listed in Table prohibitin, DDH, and CYP2A13 at 24 h showed a decrease in expression in some of the cell strains analyzed. Samples not available for RT-PCR analysis are listed as N/A in Table Real-time PCR was used to confirm and extend results seen by microarray analysis for selected genes. Following the original microarray analysis, patterns of some genes appeared to be changing at the latest time point 120 min), so extended time points were selected (12 and 24 h) to see a more complete expression profile for these genes. Due to limited amount of cDNA, genes were analyzed at 15 min and/or 120 min, and then analyzed at 12 and 24 h by RT-PCR. Extended time points were selected to look at specific genes found altered at the earlier time points. These genes were selected due to their function and/or pattern of expression, and determining their expression pattern at later time points was of interest. Results are shown in Table 0 min, soThe purpose of this study was to determine if microarray analysis of four normal human mammary cell strains with a known haplotype could be used to find biomarkers related to either exposure in general or the specific haplotype in question. The use of only four cell strains was determined to have enough power to provide basic information to lead to further study if necessary. This study would be followed up for specific genes of interest in a larger number of cell strains, preferably bypassing the more expensive and time-consuming microarray analysis for RT-PCR only.DNA microarray analysis was used to profile the cellular response to OTQ, a quinoxaline pesticide. Analysis revealed genes with common response across the four human cell strains studied as well as inter-individual variation in response. Using only four normal human cell strains, our goal was to discover any distinctly altered genes in response to OTQ exposure. Future analysis with a larger number of cell strains will be used to follow-up this analysis on specific genes of interest.The majority of studies looking at gene expression profiles have used animal models, limiting any knowledge obtained to genetically similar organisms. Analysis with normal human cell strains, like those described here, will give more information on inter-individual variation in response to various chemicals. This information will yield clues to the metabolic pathways of the specific chemicals, and this increased knowledge will aid in determining potential hazards in the environment and the workplace. Given the large number of pesticides in use today, further examination of the effect of these chemicals on individuals is warranted.junB and cfos (the AP1 complex). A number of genes involved in carcinogenesis were found altered after exposure to OTQ, both induced and down-regulated. For example, prohibitin expression is found to be up-regulated in most cell strains after OTQ exposure, with similar expression patterns associated with a decrease in cancer incidence [Following exposure to OTQ, NHMECs showed alterations in genes involved in a variety of functions. These included xenobiotic metabolism, transcription, and DNA synthesis. Genes altered as a result of OTQ exposure in all strains analyzed included transcription factors like ncidence .cytochome P4502A13 (CYP2A13) and dihydrodiol dehydrogenase, are involved in xenobiotic metabolism. It has been suggested that CYP2A13 is the main metabolic activator of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a tobacco-specific nitrosamine [CYP2A13 begins to wane at the final time point tested, suggesting this alteration to be somewhat transient. This is an unusual expression pattern for a cytochrome p450, as genes in this family tend to show a gradual increase in induction, and an equally gradual decrease in expression. Further analysis by RT-PCR showed that this p450 was increased at later time points , cyclin D1 (Z29087), and BTF2 (U72649) may further support an association between OTQ exposure, p53 variant status and carcinogenesis [GADD45 transcription in response to DNA damage, which is associated with an increase in cell cycle arrest and DNA damage repair, while increased levels of raf oncogene have been associated with lung carcinogenesis [Inter-individual variation as a result of genetic polymorphisms in genes of interest would focus on specific at-risk worker populations. For example, the four cell strains analyzed in this study have been genotyped for a variety of genes, in particular those involved in cell cycle control and xenobiotic metabolism. Two of the four cell strains selected for analysis are heterozygote for the minor variant haplotype of p53, a cell cycle control gene (cell strains 3 and 4). Although no biological mechanism for the role of this variant in carcinogenesis has been defined, several studies associating this haplotype with various cancers support such role -35. Analogenesis -38. Of togenesis -41.p53 variants in gene expression differences. Due to the expense of microarray analysis, this is performed in only a limited number of cell strains (4), and selected genes will be further analyzed with |RT-PCR in a larger number of cell strains. Over 80 cell strains have been established in our laboratory to date, with half of these having been genotyped for p53. However, given that this haplotype is found only in a limited portion of the population, this varied pattern of expression in key genes in cell cycle control may highlight a specific at-risk population.Given the small number of cell strains used, this analysis needs to be extended to additional cell strains to determine the role of the Searches for similar natural compounds to replace these potentially disruptive chemicals can also use gene expression profiles . ProfileCYP2A13 and dihydrodiol dehydrogenase. The results shown here do not suggest a direct role of OTQ in carcinogenesis. They do, however, suggest OTQ exposure leads to an increase in expression of genes that do play a direct role in carcinogen; metabolism  [The overall goal of this project was to create a gene expression profile for OTQ or related pesticide analogues with the hopes of finding genes to be used as potential biomarkers of exposure. This expression profile may also be used to determine the final role of OTQ in carcinogenesis by comparing it to profiles of known carcinogens. It is possible that the main effect of OTQ exposure is not on the direct alterations in many genes, but on alterations in genes potentially involved in carcinogenesis, among them the examples of  cancer) ,43.Discovery of genes altered following exposure to OTQ in human cell strains may aid in future epidemiology studies on pesticide exposures. Gene expression profiling can be used to yield genetic biomarkers of exposure that, after validation, could be used in a clinical setting for early determination of organophosphate exposure, increasing early treatment of pesticide illness and thereby increasing the recovery rate of exposed individuals.OTQ, oxythioquinoxDDH, dihydrodiol dehydrogenaseDMSO, dimethylsulfoxideSLR, signal log ratioDMT, Data Mining Tool™MAS, Microarray Suite™SOM, self-organizing mapNHMEC, normal human mammary epithelial cellRT-PCR, real-time polymerase chain reactionNone declared.MRG participated in the design of the study, and performed all experiments. DLW was responsible for the growth and maintenance of all cell strains used. AW conceived of the study and participated in the design and coordination. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Escherichia coli strain in which RecBCD has been genetically replaced by the bacteriophage λ Red system engages in efficient recombination between its chromosome and linear double-stranded DNA species sharing sequences with the chromosome. Previous studies of this experimental system have focused on a gene replacement-type event, in which a 3.5 kbp dsDNA consisting of the cat gene and flanking lac operon sequences recombines with the E. coli chromosome to generate a chloramphenicol-resistant Lac- recombinant. The dsDNA was delivered into the cell as part of the chromosome of a non-replicating λ vector, from which it was released by the action of a restriction endonuclease in the infected cell. This study characterizes the genetic requirements and outcomes of a variety of additional Red-promoted homologous recombination events producing Lac+ recombinants.An cat was not frequently co-inherited with cat. (5) Recombination between phage sequences in the linear DNA and cryptic prophages in the chromosome was responsible for most of the observed Lac+ recombinants. In addition, observations were made concerning recombination events between the chromosome and circular DNAs: (6) Formation of recombinants depended upon both RecA and, to a lesser extent, Red. (7) The linked tetracycline-resistance marker was frequently co-inherited in this case.A number of observations concerning recombination events between the chromosome and linear DNAs were made: (1) Formation of Lac+ and Lac- recombinants depended upon the same recombination functions. (2) High multiplicity and high chromosome copy number favored Lac+ recombinant formation. (3) The Lac+ recombinants were unstable, segregating Lac- progeny. (4) A tetracycline-resistance marker in a site of the phage chromosome distant from lac and cryptic prophage sequences in the chromosome generates a partial duplication of the bacterial chromosome. When the incoming DNA species is circular rather than linear, cointegrates are the most frequent type of recombinant.The Lac+ recombinants arise from events in which homologous recombination between the incoming linear DNA and both  Escherichia coli strain in which RecBCD has been genetically replaced by Red exhibits greatly elevated levels of recombination between its chromosome and short linear double-stranded DNA species sharing sequences with the chromosome [The Red recombination system of bacteriophage λ promotes efficient double strand break repair/recombination. An romosome .cat gene and flanking lac operon sequences recombines with the E. coli chromosome to generate a chloramphenicol-resistant Lac- recombinant [red and the bacterial recombination genes recA, recF, recO, recR, recQ, ruvAB, and ruvC. Large numbers of chloramphenicol-resistant Lac+ recombinants were generated in these crosses as well. In this study, we characterize the Lac+ recombinants and the processes which generate them. Most appear to arise from events in which homologous recombination between the incoming DNA and the chromosome generates a partial duplication of the bacterial chromosome.In previous studies, we have characterized a recombination event, pictured in Figure ombinant -4. The drecA, recF, recO, recR, recQ, ruvAB, and ruvC. Deletion of recG increased the production of both Lac+ and Lac- recombinants. Lac+ recombinants accounted for 10–60% of the total chloramphenicol-resistant offspring of the crosses. These results strongly suggest that formation of the Lac+ recombinants takes place via homologous recombination, though they do not rule out the possibility that non-homologous end joining, or other varieties of "illegitimate" recombination, might be involved as well.Data presented in Table lac::cat sequences would be attached to phage sequences. Heitman et al. [in vivo are rapidly repaired by DNA ligase. The results of previous physical studies with other substituted λ phages cut in vivo by PaeR7 lead to the expectation that the λ lac::cat819 chromosome would be uncut, or only singly cut, much of the time in the infected cell [The reason for considering mechanisms other than homologous recombination in the formation of the Lac+ recombinants is because of the expectation that some of the phage-borne n et al.  showed tted cell ,6.lacZ (not shown). When the lac::cat dsDNA was delivered into the cell by a λ vector bearing a tetracycline resistance determinant (Δ nin::tet859 – described below) neither the Lac+ nor Lac- recombinants acquired tetracycline resistance. These findings suggest that the process which formed the recombinants did not involve the entire phage chromosome.Preliminary characterization of the Lac+ recombinants formed in cells which were wild-type for all the recombination functions revealed that most, possibly all, were unstable. When streaked on plates containing chloramphenicol and X-Gal, they segregated Lac- (colorless) progeny at variable frequencies. Southern gel analysis of chromosomal DNA from unstable Lac+ recombinants revealed that some of them were multiploid for lac locus, will produce a Lac+, chloramphenicol-resistant recombinant. Data presented below, in which simpler lac::cat recombining substrates generate Lac+ recombinants at a frequency approximately 1000-fold lower than Lac-, suggest the frequency of spontaneous lac duplications in Red+ but otherwise wild type E. coli is approximately 10-3, consistent with estimates of spontaneous duplication frequency in Salmonella made by Roth et al. [A baseline level of Lac+ recombinants was to be expected in these crosses. The recombination event pictured in Figure h et al. . The Laclac::cat segment were to recombine with more than one chromosome at a time. Further complicating interpretation of the experiment, the cells were infected at a multiplicity of 10 phages per cell.The crosses described above were done by infecting log phase host cells grown in rich medium. Such cells contain multiple copies of their chromosome. Complications might arise if the lac::cat segment and single copies of the bacterial chromosome. As shown in Table To reduce the complexity of the system, we switched to a cross procedure involving low multiplicity infection (0.1 phage per cell) of stationary phase cells. Presumably, under these conditions, most of the events producing chloramphenicol-resistant recombinants take place between single copies of the lac::cat, which are diagrammed in Figure lac::cat819, the progenitor of this series, has two PaeR7 sites. Upon infecting a host cell bearing the Δ recBCD::Ptac-gam-bet-exo-pae-cI substitution, it injects its chromosome, which circularizes. Expression of its lytic genes, including those necessary for phage DNA replication, is blocked by the action of cI repressor. Cutting of its chromosome by the PaeR7 restriction endonuclease in the infected cell releases a 3.5 kbp linear dsDNA, consisting of the cat gene and 1.3 kbp flanks of lac sequences on the right and left. (In this and subsequent descriptions, "right" refers to the upstream, or 5' end of the lac operon). The ends of the flanking lac sequences match precisely their counterparts in the chromosome.To examine the dependence of Lac- and Lac+ recombinant formation on the structure of the DNA substrate, we constructed variants of λ lac::cat930 and λ lac::cat931 have only single PaeR7 sites, on the right and left, respectively. Cutting by PaeR7 in the infected cell should produce large linear dsDNAs related to the lac::cat819 fragment, but with long tails of non-homologous DNA extending from the left and right sides, respectively. Interestingly, both of these single-cut phages produce recombinants more efficiently than the double-cut λ lac::cat819, in both recG+ and Δ recG backgrounds  Some of the λ lac::cat929 chromosomes might be cut by the EcoK restriction endonuclease. The infected cells contained a functional EcoK restriction-modification system. λ lac::cat929 was grown in a similarly EcoK+ host, and therefore was presumably EcoK-modified. However, if the modification were not complete, there might be residual EcoK restriction activity, resulting in 10% of the phage chromosomes being cut. (The phage chromosome contains five EcoK sites. If each site were 98% methylated, then approximately one in ten phage chromosomes would have a single unmethylated site). (2) The production of the recombinants might not be Red-mediated. (3) The linear partner might be a broken bacterial chromosome.Approximately half of the strongly chloramphenicol-resistant recombinants generated by the uncuttable phage λ  partner . How, thhsdR, with a tetracycline resistance determinant. This replacement had little effect on the formation of recombinants by λ lac::cat929 . A fourth event, also not shown, involves recombination between λ genes and homologues in cryptic prophages in the E. coli chromosome (discussed below). As suggested in the figure, cointegrates formed by recombination in the right-side lac flank are phenotypically Lac-, while recombination in the left-side lac flank (or other loci) forms Lac+ recombinants. To test this idea, we constructed variants of λ lac::cat missing either or both lac flanks; they are diagrammed in Figure Cointegrates could in principle be formed by four different single reciprocal recombination events between the circular λ cat cassette with 1000 bp homologous flanks was only 10-fold more efficient than one with 40 bp flanks. Their experiments were done with cells which had been subjected to heat shock and electroporation. To test the efficiency of short-flank recombination under less extreme conditions, we constructed a short-flank λ lac::cat variant.The ability of the λ Red system to promote recombination events involving short sequence homologies makes it particularly useful for genetic engineering. Yu et al.  reportedlac::cat921 consists of the cat gene flanked by 40 bp sequences corresponding exactly to the terminal 40 bp at each end of the 1.3 kbp flanks of λ lac::cat819. As shown in Table lac::cat921 exhibits a several hundred-fold lower efficiency of recombinant formation than its long-flank counterpart, in both recG+ and Δ recG backgrounds. λ lac::cat921 was similarly inefficient in log phase cells (data not shown). These observations suggest that short-flank recombination may not be a significant activity of the Red system in nature. However, they do not speak to the question of whether long flanks work better because they provide a larger homology target for synapsis, or because they provide non sequence-specific protection, perhaps delaying exonucleolytic degradation of the recombining sequences long enough to permit recombination to take place.The fragment released by PaeR7 from the chromosome of λ lac::cat930, but not by the left side-cut λ lac::cat931  cloning the cat gene from the deletion mutant, along with flanking chromosomal sequences; (3) using the cloned, plasmid-borne flanking sequences, and Red-mediated gap repair, to clone the lac operon in a plasmid; (4) inverting the lac operon, relative to its flanking sequences, in the plasmid; (5) replacing the Δ lac::cat chromosomal allele with the plasmid-borne lac-inv allele.The strategy for inverting the chromosomal lac::cat930 and lac::cat931 alleles were constructed by replacing the left-side and right-side PaeR7 sites, respectively, of pTP819, with XbaI sites. To produce inverted derivatives of these two alleles, the plasmids pTP930 and pTP931 were digested with XbaI and XhoI (a PaeR7 isoschizomer); lac::cat inserts and bla-ori backbone fragments from the two plasmids were exchanged. The inverted alleles were then crossed into phage λ, as described in the Methods section.The single-cut E. coli K-12 [lac::cat930 and λ lac::cat931, and the efficiency of Lac+ recombinant formation by λ lac::cat930, were slightly elevated relative to those in the corresponding AB1157-derived strain.In constructing the chromosomal inversion, we switched genetic backgrounds, from the AB1157-derived strains with which most recombination studies have been done, to MG1655, the sequenced wild type oli K-12 . In the lac-inverted E. coli) were tested for Lac+ recombinant formation. The results are shown in Figure lac::cat930  in lac-wild type, and lac::cat1033 (left-cut, inverted orientation) in lac-inv.The eight combinations of normal and inverted phages and bacteria  lacking its sequences derived from phage P22 was equally proficient at generating Lac+ recombinants. A derivative missing the left lac flank made Lac+ recombinants almost exclusively; the small number of Lac- recombinants in this case probably represent cointegrates made by uncut plasmid DNA in the fragment preparation.We constructed several other linear DNA substrates to test the generality of the model in Figure ion  and λ lac::cat930 (cut right) behave like their counterparts generated by electroporation of linear dsDNAs into Red+ cells: they segregate Lac- chloramphenicol-resistant clones at low frequency, and Lac+ chloramphenicol-sensitives at high frequency (data not shown). In addition, their formation also depends upon the presence of cryptic prophages in the chromosome: λ lac::cat819 was found to produce Lac+ chloramphenicol-resistant recombinants as only 0.13% (average of six measurements) of the total chloramphenicol-resistant progeny in crosses with TP750. These properties suggested both kinds of recombinant might have the same structures as well, but PCR tests of 50 of the phage-generated recombinants with the primers used to demonstrate type I, II, and III recombinants  when electroporated directly into cells .One aspect of Lac+ recombinant formation by λ ls Table . How doelac::cat-bearing λ phages and the bacterial chromosome, as well as the mechanism of their formation. Our studies uncovered a diversity of recombinant structures, including complex duplications and cointegrates. Recombination between λ and cryptic prophage sequences in the bacterial chromosome was found to be the most significant mechanism generating Lac+ recombinants. All of the Lac+ recombinants could be generated by homologous recombination events of types which have been previously described. In particular, there was no evidence for end-joining or other non-homologous recombination events. Perhaps the most surprising finding was the high frequency with which large duplications in the bacterial chromosome – up to 1 Mbp – could be generated by the Red system.The bacteriophage λ Red recombination system is of general interest for two main reasons. First, it is an intensively characterized and relatively simple system, which serves as a model for studies of homologous recombination -17. SecoE. coli strain DH5α (λ pir) [hsdR::tet allele in TP842 was constructed by using a Tn 10-containing E. coli strain as template in PCR with primers hsdRut (5'-TTGGACAGGCCCGCACAGCAATGGATTAATAACAATGATGCTCGACATCTTGGTTACCGT-3') and hsdRdt (5'-GCTGAATTTGCCCAGCAGGGTATCGAGATTATCGTCAAAGCGCGGAATAACATCATTTGG-3'). The Δ lac::cat allele in TP872 was constructed by using a Tn 9-containing E. coli strain as template in PCR with primers cat15 (5'-TCTGGTGGCCGGAAGGCGAAGCGGCATGCATTTACGTTGAATGAGACGTTGATCGGCACG-3') and cat16 (5'-AGAGTACATCTCGCCGTTTTTTCTCAATTCATGGTGTACAATTCAGGCGTAGCACCAGGC-3'). (λ pir)  was usedcI857 Sam7 containing the nin region genes was cloned into the EcoR1 site of pBR322. In the resulting plasmid, pnin, the nin genes are read clockwise in the conventional map of pBR322. pTP772 was constructed by deleting sequences between the two HindIII sites of pnin. pTP859 was constructed by cutting pTP772 with SacII and ClaI, blunting the ends with T4 DNA polymerase, ligating with NotI linkers (5'-AGCGGCCGCT-3'), cutting with NotI, and ligating with a NotI fragment of pTP857 [tetR and tetA genes of transposon Tn10.An EcoR1 fragment of λ f pTP857  containicat gene is flanked on both sides by lac sequences, PaeR7 sites, and λ sequences (for crossing into the phage), has been described [lac flank and PaeR7 site. pTP922 was constructed by deleting the lac and cat sequences between the two PaeR7 sites in pTP819. (In this and other plasmid constructions, PaeR7 sites were cut with XhoI, an isoschizomer). pTP926, pTP927, and pTP928 were constructed by ligating the oligonucleotide 5'-TCGACAGTCTAGACTG-3' into the PaeR7 sites of pTP828, pTP829, and pTP922, respectively, eliminating the PaeR7 sites and replacing them with XbaI sites. pTP929 was constructed by ligating the XbaI-NcoI fragment of pTP926 containing the N-terminal coding sequences of cat and the NcoI-XbaI fragment of pTP927 containing the C-terminal coding sequence of cat into the XbaI site of pTP928. The orientation of the reconstructed cat gene is the same as in pTP819. pTP930 was constructed by ligating together the large ApaI-SacII fragment of pTP929 and the small ApaI-SacII fragment of pTP819. pTP931 was constructed by ligating together the small ApaI-SacII fragment of pTP929 and the large ApaI-SacII fragment of pTP819.pTP819, in which the escribed . pTP828 cat gene from a Tn9-containing E. coli strain with primers 5'-GACGCACTCGAGGCGTTAACCGTCACGAGCATCATCCTCTGCATGGTCAGGCCGGCCACTGGAGCACCTCAAAAACACCA-3' and 5'-GACGCACTCGAGGCACACAGCGCCCAGCCAACACAGCCAAACATCCGCGCGGGCCCGACCGGGTCGAATTTGCTTTCGAA-3'. The presence of the expected sequences from the synthetic oligonucleotides in pTP921 DNA was verified by automated sequencing (data not shown).pTP921 was constructed by ligating together two XhoI-digested DNAs: pTP922 and a PCR product made by amplifying the lac flank only) was constructed by replacing the XbaI-ApaI lacZY segment of pTP930 with a mixture of two oligonucleotides, 5'-CTAGTTGCAAGCTTGGGCC-3' and 5'-CAAGCTTGCAA-3'. pTP989 (right lac flank only) was constructed by replacing the NgoMI-XhoI lacZ segment of pTP930 with a mixture of two oligonucleotides, 5'-CCGGCAAGCTTGCTGGTGGGCAA-3' and 5'-TCGATTGCCACCAGCAAGCTTG-3'. pTP995 (no lac flank) was constructed by ligating together the small NcoI fragment of pTP988 and the large, ori-containing NcoI fragment of pTP989.pTP988 (left cat-containing fragment of pTP930 and the XbaI- and PaeR7-ended ori-containing fragment of pTP931. pTP1033 was constructed by ligating together the XbaI- and PaeR7-ended cat-containing fragment of pTP931 and the XbaI- and PaeR7-ended ori-containing fragment of pTP930.pTP1032 was constructed by ligating together the XbaI- and PaeR7-ended cat gene and flanking sequences from strain TP872 (Δ lac::cat) with primers 5'-CATCATCACGCGGCCGCGACGTTTGCCGCTTCTGAA-3' and 5'-ATCATCCACGCGGCCGCTGCGTTTTGCACCAGTACG-3'. In pTP1016, the cat gene is flanked closely by unique SphI and BsrGI sites. pTP1027 was constructed by electroporating SphI- and BsrGI-digested pTP1016 into strain TP829 (lac+); a plasmid formed by gap repair was isolated. pTP1028 was constructed by ligating SphI-digested pTP1027 with the oligonucleotide 5'-GTTGTACAACCATG-3', converting the SphI site into a BsrGI site. pTP1034 was constructed by cutting pTP1028 with BsrGI, ligating the two fragments back together, and screening for plasmids in which the lac genes were inverted relative to their flanking sequences.pTP1016 was constructed by ligating into the NotI site of pTP809  the NotIpir function, was constructed by ligating together two AatII- and Bam-digested DNA species: a PCR product made by amplifying a Tn10-containing E. coli strain with primers 5'-TCAACGTAAATGCATGGACGTCCTCGACATCTTGGTTACCGT-3' and 5'-TGTACACCATGAATTGGATCCCGCGGAATAACATCATTTGG-3'; and the ori-containing fragment of plasmid pLD54 [lac::cat-containing fragments of pTP819, 930, 931, 1032, and 1033, respectively, into the BamHI site of pTP1029.pTP1029, a tetracycline resistance bearing vector capable of replicating only in cells expressing R6K id pLD54 . pTP1018cat-containing fragment of pTP1051 into the BamHI site of pTP1029.pTP1047 was constructed by ligating the complementary oligonucleotides 5'-TCGTCTAGAGT-3' and 5'-CCGGACTCTAGACGAAGCT-3' between the SacI and NgoMIV sites of pTP1019. pTP1051 was constructed by ligating the complementary oligonucleotides 5'-CAGCATGCAT-3' and 5'-CTAGATGCATGCTGGTAC-3' between the KpnI and XbaI sites of pTP930. pTP1052 was constructed by ligating the complementary oligonucleotides 5'-CAGCATGCAGGGCC-3' and 5'-CTGCATGCTGGTAC-3' between the KpnI and ApaI sites of pTP1019. pTP1053 was constructed by ligating the complementary oligonucleotides 5'-GGCATGCAGGTTCTTTGAGTCCTTTGGGCGGCCGCGGGCC-3' and 5'-CGCGGCCGCCCAAAGGACTCAAAGAACCTGCATGCCGC-3' between the SacII and ApaI sites of pTP1019. pTP1054 was constructed by ligating the complementary oligonucleotides 5'-CCGGCGCGGCCGCAGGTTCTTTGAGTCCTTTGGTGTACAGAGCT-3' and 5'-CTGTACACCAAAGGACTCAAAGAACCTGCGGCCGCG-3' between the NgoMIV and SacI sites of pTP1020. pTP1055 was constructed by ligating the small SphI-NotI fragment of pTP1016 between the SphI and NotI sites of pTP1053. pTP1056 was constructed by ligating the small NotI-BsrGI fragment of pTP1016 between the NotI and BsrGI sites of pTP1054. pTP1057 was constructed by ligating the BamHI Ptac-gam-bet-exo-pae-cI operon flanked by sequences upstream from recC on one side and sequences internal to recD on the other, has been described [cI gene from E. coli strain TP507 with the same primers used in the construction of pTPP822. This construction has the effect of simply deleting the pae genes from pTP822. pTP996 was constructed by deleting the C-terminal coding sequences of bet and the N-terminal coding sequences of exo between the two HpaI sites of pTP978. pTP979 was constructed by ligating together two NcoI- and XbaI-digested DNAs: pTP822 and a PCR product made by amplifying the bla gene from pBR322 with primers 5'-CCACCAATCATCCATGGCGCGGAACCCCTATTTGTTT-3' and 5'-TTGTTGGACGATCTAGAGGTCTGACAGTTACCAATGC-3'. This construction has the effect of replacing pae and cI with bla.pTP822, which bears a synthetic lac::cat819 and λ lac::cat819 nin5 have been described [nin::tet859 was made by crossing λ wild type with plasmid pTP859, infecting a tetracycline-sensitive strain with the resulting lysate, and selecting a tetracycline-resistant lysogen. The Δ nin::tet859 substitution replaces λ bp 40388–43825 with the tetR and tetA genes of transposon Tn 10.λ escribed . λΔ nin:lac::cat921, 929, 930, 931, 988, 989, 995, 1032, and 1033 substitutions were crossed into λ wild type and/or λ Δ nin::tet859. The parent phages were crossed with the cat substitution-bearing plasmids. Phages which had acquired the substitution were either selected by plating on a strain lysogenic for phage P2 (Spi- phenotype), or identified by their clear-plaque morphologies . The structures of the substituted phages were all verified by their ability to generate specific products when used as templates in PCR (not shown).The 4), and shaken with chloroform. Two methods for crosses monitoring recombination between phage-injected DNA and the bacterial chromosome, both previously described, were employed: high-multiplicity infection of log phase cultures [Phages were crossed with plasmids by spotting enough phage to make a confluent zone of lysis on a lawn of a sensitive bacterial strain bearing the parent plasmid. After overnight incubation at 37°C, material from the zone was collected in TM  dimethylsulfoxide, 20 mM Tris-HCl pH 8.4, 50 mM KCl, and primers at 0.7 μM. Samples were heated to 95C for 5 min, then put through 30 cycles of 94C for 1 min, 55C for 1 min, 72C for 2–4 min, depending upon the length of the expected products. Primers for demonstrating the type I, II, and III tfa junctions were combined in a single mixture of 4 oligonucleotides: Jsp1 (GTTGAATGGGCGGATGCTAA), Jsp2 (TCTTCCACCAGAAAGCTACC), JncA (TGCCGTGTGAACGGTTTAC), and Jnc2 (TTCTAGCCCCATCATCTGTG). Primers for spanning the ACF-generated duplication  and Csj2 (CGAATAGTCGGCTCAACGTGGGTT).The structures of various Red-generated duplications in the PCR: polymerase chain reaction. X-Gal: 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside. IPTG: isopropyl β-D-thiogalactopyranoside. bp: base pairs.ACF carried out the experiments summarized in Table


HTLV-1 is the etiological agent of adult T-cell leukemia (ATL), the neurological syndrome TSP/HAM and certain other clinical disorders. The viral Tax protein is considered to play a central role in the process leading to ATL. Tax modulates the expression of many viral and cellular genes through the CREB/ATF-, SRF- and NF-κB-associated pathways. In addition, Tax employs the CBP/p300 and p/CAF co-activators for implementing the full transcriptional activation competence of each of these pathways. Tax also affects the function of various other regulatory proteins by direct protein-protein interaction. Through these activities Tax sets the infected T-cells into continuous uncontrolled replication and destabilizes their genome by interfering with the function of telomerase and topoisomerase-I and by inhibiting DNA repair. Furthermore, Tax prevents cell cycle arrest and apoptosis that would otherwise be induced by the unrepaired DNA damage and enables, thereby, accumulation of mutations that can contribute to the leukemogenic process. Together, these capacities render Tax highly oncogenic as reflected by its ability to transform rodent fibroblasts and primary human T-cells and to induce tumors in transgenic mice. In this article we discuss these effects of Tax and their apparent contribution to the HTLV-1 associated leukemogenic process. Notably, however, shortly after infection the virus enters into a latent state, in which viral gene expression is low in most of the HTLV-1 carriers' infected T-cells and so is the level of Tax protein, although rare infected cells may still display high viral RNA. This low Tax level is evidently insufficient for exerting its multiple oncogenic effects. Therefore, we propose that the latent virus must be activated, at least temporarily, in order to elevate Tax to its effective level and that during this transient activation state the infected cells may acquire some oncogenic mutations which can enable them to further progress towards ATL even if the activated virus is re-suppressed after a while. We conclude this review by outlining an hypothetical flow of events from the initial virus infection up to the ultimate ATL development and comment on the risk factors leading to ATL development in some people and to TSP/HAM in others. In cu T-cells -6, resul T-cells . Clonal patients . CD8+ T-infected , but theinfected .Shortly after infection the virus enters into a latent state, rendering the infected individuals asymptomatic seropositive carriers. About 5% of these individuals develop one of the viral associated diseases 10 to 40 years after infection. During latency the viral gene expression in the peripheral blood lymphocytes (PBLs) of such carriers is very low. Viral RNA is undetectable by Northern blot analysis in most of the infected cells  freshly isolated from their peripheral blood . FurtherAlthough the mechanism of HTLV-1 pathogenicity is not fully understood yet, it is widely believed that a virally encoded transactivator protein, called Tax, plays a central role in this mechanism. It should, therefore, be noted that while the low level of the virus gene expression detected in latently infected carriers might be sufficient for maintaining their anti HTLV-1 seropositivity and CTL activity, the low Tax level, resulting from this reduced viral expression is, most likely, below its pathogenic threshold. This implies that generating an HTLV-1 related disease requires an activation of the dormant virus in order to elevate Tax to its pathogenic level.In this article we present a comprehensive review of the wide range of Tax molecular interactions and biological effects that might be closely relevant to the mechanism of ATL genesis and summarize this information by proposing hypothetical flow of a stepwise pathway leading to this malignancy or to TSP/HAM.HTLV-1 is a complex retrovirus that, in addition to the two long terminal repeats (LTRs) and the gag, protease, pol and env genes, which are typical to most other retroviruses, its genome contains an additional region called pX, which resides between the env gene and the 3'-LTR,. This region includes four partially overlapping reading frames (ORFs), of which the most investigated ones are ORFs III and IV that encode for the viral regulatory Rex and Tax proteins respectively  residing at its amino terminus ,21. Howe unclear .More recently interest has been focused also on ORF I that encodes for p12 and p27 and ORF II that encodes for p13 and p30 proteins . In contThe frame II encoded p30 protein has been shown to localize to the nucleus and to function as a transcription factor. Transient transfection experiments have demonstrated that this protein can modulate the expression of various promoters and to activate HTLV-I LTR expression independently of Tax . It was Fig. Since Tax protein is widely regarded as a key element in the HTLV-1 related leukemogenic process. We will discuss in the following sections the molecular activities and biological effects of Tax that seem to contribute to its oncogenic potential.As noted before, Tax was initially discovered as a transactivator of the HTLV-1 gene expression . It actiOf particular note are also the recent observations that when two copies of the TxRE are placed upstream to TATAA boxes from HTLV-1 LTR or from other promoters, the strongest activation by Tax is detected with the TATAA box of the HTLV-1 LTR, indicating that this TATAA box contains a specific Tax responsive element. Furthermore, these studies have also revealed that beside of the enhancing effect Tax on the association of the TATAA-box binding protein (TBP) to the TATAA site, Tax has an additional stimulatory effect that is directed towards a step occurring after the assembly of the basal transcriptional factors onto the TATAA box .Many cellular genes contain in their promoters a consensus CRE element and are activated by signals that elevate the cellular cAMP level. The elevated cAMP activates PKA to phosphorylate CREB which, in turn, binds to CRE and to CBP/p300. However, there is a substantial controversy on whether Tax can activate only the viral CRE in its context with the CG-rich flanking domains in the viral LTR ,61, or a6GG], and an upstream Ets box [GGA(A/T)]. After binding to the CArG box, SRF protein interacts with the ternary complex factors (TCFs), which consequently bind to the upstream Ets box. In addition, SRF requires for its transcriptional activity the CBP/p300 and p/CAF co-activators [HTLV-1 infected and Tax-expressing T-cell lines display increased expression of immediate early genes such as c-Fos, c-Jun, JunB, JunD and Fra-1, which are components of the dimeric transcription factors AP1, Egr-1 and Egr-2 , fra-1 , Krox-20tivators .Tax activates these immediate early genes by interacting with SRF ,78 and wA substantial part of Tax oncogenic potential is attributed to its ability to activate transcription factors of the NF-κB family, since these factors regulate the expression of numerous cellular genes  associatIn non-activated state NF-κB factors are trapped in the cytoplasm, tightly associated with inhibitory proteins called IκBs, primarily with IκBα and IκBβ. These inhibitors contain ankyrin repeats through which they bind to the RHD of the NF-κB factors and mask their nuclear localization signal (NLS) . In addi on serine19 and serine23  [ share a 52% amino acid identity and a similar domain structure that includes amino-terminal kinase domain, a dimerization leucine zipper domain, and helix-loop-helix motifs, which are involved in regulating their kinase activity [The cytoplasmic phase includes phosphorylation of IκBα on serine32 and serine36 and of IκBr (NEMO) ,90  Fig. , No. 2b,erine181 ,94. Desperine181 ,95,96. Terine181 ,98  can bind the CBP/p300 and P/CAF coactivators which are essential for the transcriptional competence of p65(RelA):p65(RelA) and p65(RelA):p50 dimers . This bi and maintains, in this manner, a persistent NF-κB transcriptional activation [In addition to its cytoplasmic inhibitory function IκBα plays an important regulatory role in the nucleus too. IκBα has an NLS signal which enables its translocation to the nucleus where it is protected from the signal-induced degradation described above . Within tivation . has also been found to have an important role in the nucleus  and IKKα for CBP/p300   The most widely accepted concept is that Tax associates with the IKK complex through the adaptor IKKγ/NEMO subunit. Tax also binds to the upstream kinases, MEKK1 and NIK and enhances their kinase activity. In this manner Tax connects these activated kinases to IKKγ/NEMO and recruits their kinase activity to phosphorylate IKK and IKKβ [b) Tax can bind directly to IKK and IKK and activates their kinase activity independently of their phosphorylation by the upstream signal-induced kinases [c) Tax can bind directly to the IκBs and induce their proteosomal degradation independently of their phosphorylation by IKK [In contrast to the transient NF-κB activation by external signals, NF-κB factors are constitutively activated by HTLV-1 Tax protein in Tax-expressing and HTLV-1-infected cells. Reported studies suggest that Tax may exert this activation in three ways: and IKKβ ,120-122 and IKKβ . b) Tax  kinases  Fig. 3,,a The mon by IKK ,125  is involved in DNA synthesis and maintenance of the genome stability by participating in DNA repair and chromosome condensation. It alters DNA topology by transiently breaking one strand of the DNA, passing the other strand through the break and finally resealing the break . Tax hasAll the above effects of Tax which enhance mutations and other chromosomal aberrations and interfere with DNA repair should be expected to induce cell cycle arrest or apoptosis. This, in turn, should prevent further progression of the leukemogenic process in the infected cells, unless such cells can, somehow, escape the cell cycle arrest and apoptosis. There is a substantial controversy over the influence of Tax on the cell response to stress insults. While many studies have demonstrated that Tax protects cells from stress-induced cell cycle arrest or apoptosis -186, othNumerous studies have been focused on investigating Tax oncogenic potential in cultured cells and animal models. Most of them used plasmids expressing w.t. Tax or various Tax mutants under the control of HTLV-1 LTR or other different promoters. It was only few years ago that an infectious clone of the entire HTLV-1 genome was constructed and appropriate cell culture techniques were developed to introduce this clone into human primary PBLs. This clone was found as capable of propagating in cultured mammalian cells and to transform primary human T-lymphocytes -197. ThiWAF-1 which prevents apoptosis and enhances the replication of the transformed cells [Tax has been shown to induce neoplastic transformation of the rat fibroblast Rat-1 -203 and ed cells . The cooed cells . It shoued cells . Therefoed cells ,153,165.WAF-1 [A closer insight into the ATL leukemogenesis has been gained through studies using primary human T-lymphocytes. Such experiments have revealed that after infection in culture with HTLV-1 or permanent transfection with Tax, primary human T-cells undergo two stages of cellular changes. In the first stage the cell become immortalized but still remain dependent on IL-2 for their growth . This imWAF-1  and to bWAF-1 . StudiesWAF-1 ,208, othWAF-1 . On the WAF-1 . In addiWAF-1 ,210.In the second stage few IL-2-independent clones of transformed cell emerge. Such transformed cells display an IL-2-independent constitutive activation of the IL-2 receptor (IL-2R) signaling pathway that includes the Janus kinases JAK1 and JAK3, and the signal transducers and activators of transcription STAT3 and STAT5, which are constitutively active in such cells ,211-213.Many of the above changes observed in the HTLV-1/Tax immortalized and transformed primary human T-cells are quite analogous to those found in ATL cells. However, while fresh leukemic cells from ATL patients, as well as cell lines derived from these leukemic cells, are successfully engrafted in SCID mice and their leukemic infiltration to various organs is similar to that seen in ATL patients , primaryTax transgenic mice have been widely used as models for investigating the oncogenic effects of Tax in-vivo, hoping to get closer insight to the ATL leukemogenic process in human . A wide Various modes of tumor induction by the transgenic Tax have been noted so far. Hall et al.  have shoAs noted before, continuous Tax expression is required for maintaining the neoplastic phenotype of Rat-1 cells transformed by Tax in culture . In contINK4B and p16INK4A [KIP1 [In view of the above described pleiotropic effects of Tax, which are summarized in Fig. p16INK4A , p27KIP14A [KIP1 , p16  prepared the Fig.ures and together with author 2 (S-K.Y) covered the cited publications and prepared the draft of this review. Author 3 (A.M) designed the outlines of the review and together with the other two authors prepared the final version for submission. All authors read and approved the final manuscript.