False-positive TUNEL staining observed in SV40 based transgenic murine prostate cancer models.

The TRAMP (Transgenic Adenocarcinoma of the Mouse Prostate) and LADY (Probasin-large T antigen transgenic mouse) mice are widely used autochthonous models of prostate cancer. Both models utilise probasin promoters to direct androgen-regulated expression of oncogenic SV40 specifically to epithelial cells of the mouse prostate. The oncogenic processes and phenotypes which result mimic many features of human prostate cancer, making these transgenic mouse models useful experimental systems. The terminal deoxynucleotidyl transferase (Tdt)-mediated dUTP in situ nick end labelling (TUNEL) assay is a commonly used method for the detection of cells undergoing apoptosis. In this study, we demonstrate false-positive TUNEL staining in frozen prostate tissue from TRAMP and LADY mice, which was not observed in non-transgenic control animals and is not due to non-specific binding of labelled-dUTP substrate. The false-positive signal co-localised with large SV40 T-antigen expression. False-positive signal was apparent using multiple commercial TUNEL kits with different detection systems. These results caution against the use of the TUNEL assay for detection of apoptosis in frozen prostate tissue of large T-antigen based autochthonous transgenic models of prostate cancer.

Evaluation of external operculum loop tags to individually identify cage-cultured Atlantic halibut Hippoglossus hippoglossus in commercial research trials.

The growth, survival and tag retention of double-tagged [external FT4 lock-on (FT4) and internal passive integrated transponder (PIT)-tagged] Atlantic halibut Hippoglossus hippoglossus were compared to internal PIT-tagged controls in a randomized trial. The objective was to assess the suitability of these tags for monitoring the performance of individual fish in longitudinal trials under commercial cage-culture conditions in the lower Bay of Fundy, New Brunswick, Canada. The FT4 tags were chosen due to their similarity to tags used by investigators to track H. hippoglossus in the wild. A subset of the population randomly received an external FT4 tag inserted through the operculum and were monitored over a 1105 day period. The specific growth rate of FT4-tagged fish was significantly reduced in the first sea summer with no significant difference observed for the remainder of the trial. The differential growth in the first sea summer created a relative size advantage, permitting controls to increase in size significantly faster than FT4 fish in all subsequent periods. The FT4 tags did not significantly influence survival under normal commercial cage-culture conditions. Results, however, suggest that the survival of FT4-tagged H. hippoglossus may be compromised during stressful handling events. Tag retention of FT4 tags was acceptable with 76% of tags remaining at the end of the 1105 day trial. FT4 tags proved to be an effective method to identify individual H. hippoglossus, with the caveat that they seriously bias productivity measures in commercial research trials.

Early resistance to therapy during induction in childhood acute lymphoblastic leukemia.

Many patients with acute lymphoblastic leukemia (ALL) are not cured by current therapy because of the development of drug resistance. It is not clear when resistance develops during the growth of the leukemic clone and whether resistant cells are already present at diagnosis or develop later during treatment. Twenty-two uniformly treated children with ALL were studied throughout induction treatment. The size of the leukemic clone in blood and marrow was estimated by limiting dilution PCR analysis, using the rearranged immunoglobulin heavy chain gene as a molecular marker. The decline in the number of leukemic cells was biphasic in virtually all patients. For both marrow and blood, the logarithmic mean of the number of leukemic cells fell by approximately four orders of magnitude during the first 2 weeks, one order of magnitude during the third week, and not at all during the last two weeks of induction treatment. For marrow, the median of the fraction of leukemic cells in each patient that survived per week of treatment was 0.008 for the first 2 weeks, 0.12 for the third week, and 1.4 for the last 2 weeks; for blood, the corresponding figures were 0.003, 0.14, and 0.69, respectively. In individual patients, the results for marrow and blood showed good correlation. The biphasic decline of leukemic cell number suggests that most leukemic cells were sensitive to treatment and were rapidly killed, leaving behind a minor but substantial population of drug-resistant cells. The most likely explanation for this phenomenon is that these resistant cells were already present at diagnosis, their resistance having originated from genetic or epigenetic mutations during prior growth of the leukemic clone.

Prognostic significance of detection of monoclonality in remission marrow in acute lymphoblastic leukemia in childhood. Australian and New Zealand Children's Cancer Study Group.

Techniques based on the polymerase chain reaction (PCR) to detect rearrangement of the immunoglobulin or T-cell receptor genes can detect residual disease in leukemia and hence have the potential to improve prognosis and treatment. Such techniques may involve either detection of monoclonality, which is simple and quick but has limited sensitivity, or specific detection of the leukaemic clone, which is complex and time-consuming but has high sensitivity. The PCR was used to detect monoclonal rearrangements of the immunoglobulin heavy chain and/or T-cell receptor gamma chain genes in archival marrow specimens from 185 children with acute lymphoblastic leukemia who achieved remission during two consecutive Australasian trials of treatment. A monoclonal rearrangement was detected at diagnosis in 152 (84%) patients and in these patients detection of the same rearrangement in the remission marrow at the end of induction therapy was highly significantly correlated with outcome. There were nine patients in whom polymerase chain reaction showed only the monoclonal rearrangement and eight (89%) relapsed; there were 26 patients in whom PCR showed the leukemic monoclonal rearrangement as well as polyclonal rearrangements from normal lymphocytes and 12 (46%) relapsed; and there were 117 patients in whom only polyclonal rearrangements could be detected and only 29 (25%) relapsed. In patients who relapsed, remissions were shorter in those patients in whom the leukemic rearrangements had been detected in the remission marrow. Treatment in the later trial was more intensive than in the earlier trial, the results were better and the PCR detected the leukemic rearrangement in the remission marrow in significantly fewer patients. We conclude that detection by PCR of the monoclonal gene rearrangement of the leukemic clone in remission marrow indicates that numerous leukemic cells have survived induction therapy and is a good predictor of relapse. However, due to limited sensitivity of the test, failure to detect the leukemic clone by PCR is not a sufficiently good predictor of ultimate cure.

Characterization of clonal immunoglobulin heavy chain and I cell receptor gamma gene rearrangements during progression of childhood acute lymphoblastic leukemia.

Instability of antigen receptor gene rearrangements during progression of acute lymphoblastic leukemia (ALL) has important implications for polymerase chain reaction (PCR)-based techniques using these genes for the detection of minimal residual disease (MRD). Antigen receptor gene instability may lead to false negative results in bone marrow samples taken during remission. Utilizing the PCR and consensus primers for rearranged immunoglobulin heavy chain (IgH) and T cell receptor gamma (TCR gamma) gene sequences, we analyzed the bone marrow samples at diagnosis and first relapse for 37 children with ALL. The incidence of clonal evolution at the IgH locus was 9/33 (27%) and at the TCR gamma locus 1/15 (7%). In four of the nine patients with clonal evolution at the IgH locus, the sequence at relapse retained the diversity and joining region (D-N-J) sequences from diagnosis. Patients with clonal evolution were characterized by a higher incidence of more than one IgH PCR band at diagnosis and by late relapse (> 18 months from diagnosis). These results suggest that, where possible, patients with more than one IgH PCR rearrangement at diagnosis should be monitored using another antigen receptor gene, such as TCR gamma, since evolution for this gene was found to be a rare event. By combining this approach with a strategy directed at the more stable D-N-J region of the IgH gene, MRD false negativity would have occurred in less than 10% of patients in the present study.

The use of monoclonal gene rearrangement for detection of minimal residual disease in acute lymphoblastic leukemia of childhood.

Sensitive quantification of minimal residual disease (MRD) using the polymerase chain reaction (PCR) is strongly predictive of outcome in childhood acute lymphoblastic leukemia (ALL), with MRD levels at the end of induction therapy of >10(-3) predicting a poor outcome. Methods for sensitive quantification are, however, complicated and time-consuming. Detection by PCR of monoclonal immunoglobulin heavy chain (IgH) and T cell receptor (TCR) gene rearrangements is simple and can be used in routine laboratories but is non-quantitative and of lower but uncertain sensitivity. The aim of this study was to determine the value of detection of monoclonality in identification of different levels of MRD. We looked for monoclonality in 64 bone marrow aspirates which had been obtained from 31 patients with B lineage ALL at various times during induction therapy and for which levels of MRD had been determined by limiting dilution analysis using patient-specific PCR primers. Detection of monoclonality identified levels of MRD of > or =10(-3) during induction with a sensitivity of 78% and a specificity of 93%. The positive and negative predictive values were 0.86 and 0.88, respectively. The sensitivity of detection of a monoclonal IgH rearrangement was greater than that for the TCRgamma locus during induction as an IgH rearrangement was detected more often than a TCRgamma rearrangement in patients who had both IgH and TCRgamma rearrangement at diagnosis. Detection of monoclonality is therefore a simple and quick test applicable to the majority of patients with ALL and it may be useful in identifying high-risk patients at the end of induction and in identifying relapsing patients later during therapy.

Comparison of methods for assessment of minimal residual disease in childhood B-lineage acute lymphoblastic leukemia.

The level of minimal residual disease (MRD) early in treatment of acute lymphoblastic leukemia (ALL) strongly predicts the risk of marrow relapse. As a variety of methods of varying complexity have been separately used for detecting and quantifying MRD, we compared the prognostic utility of three methods measurement of blast percentage on day 14 of treatment, detection of monoclonality on day 14 or day 35, and measurement of MRD by PCR-based limiting dilution analysis on day 14 or day 35. The study group comprised 38 children aged 1-15 with Philadelphia-negative B-lineage ALL who were uniformly treated and followed until relapse or for a minimum of 5 years. We also studied some of the technical factors which influence the ability to detect MRD. Measurement of blast percentage on day 14 by an expert morphologist, detection of monoclonality on day 35, and PCR-based measurement of MRD levels on days 14 and 35 all showed significant ability to divide patients into prognostic groups. Measurement of blast percentage on day 14 by routine morphology or detection of monoclonality on day 14 were not useful. The quality of DNA samples varied greatly, as determined by amplifiability in the PCR. However, virtually all amplifiable leukemic targets in a sample were detectable which suggests that the level of detection achieved by limiting dilution analysis is essentially determined by the amount of DNA which it is practicable to study. We conclude that quantification of MRD at the end of induction provides the full range of prognostic information for marrow relapse but is complex; detection of monoclonality on day 35 is simple and has good positive predictive value; and quantification of MRD on day 14 merits further study. PCR-based methods for measurement of MRD levels should incorporate a correction for variation in DNA amplifiability.

Effect of the Philadelphia chromosome on minimal residual disease in acute lymphoblastic leukemia.

The Philadelphia translocation is associated with a poor prognosis in adults and children with acute lymphoblastic leukemia, even though the majority of patients achieve remission. To test the hypothesis that the translocation leads to drug resistance in vivo, we studied 61 children and 20 adults with acute lymphoblastic leukemia and used the level of minimal residual disease at the end of induction as the measure of drug resistance in vivo. In children the presence of the translocation was associated with a significant increase in residual disease, indicating higher drug resistance in vivo; five of seven Philadelphia-positive children but only five of 54 Philadelphia-negative children had a minimal residual disease level >10(-3), a level which is associated with a high risk of relapse in childhood acute lymphoblastic leukemia of standard risk. By contrast, in adults, residual disease and hence drug resistance was already higher than in children, and the presence of the Philadelphia translocation in seven patients had no obvious additional effect. We conclude that the Philadelphia chromosome may increase resistance to drugs in vivo in children, but not detectably in adults.

Elevated stromal chondroitin sulfate glycosaminoglycan predicts progression in early-stage prostate cancer.

Curative therapies for clinically localized prostate cancer have significant morbidity, and those patients who might be cured by aggressive management are not easily identified using current clinical information. Better biomarkers of tumor behavior need to be identified to improve clinical outcome. Chondroitin sulfate (CS), a glycosaminoglycan, may be a potentially useful biomarker as it is known to influence cell growth and differentiation and might influence malignant progression. In this study, CS was immuno-localized to the periacinar and peritumoral fibromuscular stromal tissue of nonmalignant and malignant prostates. The CS concentration was increased in the prostate tissue of men with early-stage prostate cancer compared with tissue from men without cancer (P < 0.0001). Using Cox's univariate analysis, CS concentration, tumor grade, preoperative serum prostate-specific antigen (PSA), extracapsular extension of disease, positive surgical margins, and patient age were associated with an increased risk of PSA failure. The CS concentration was compared with the other features in two-variable regression analyses. CS and preoperative serum PSA concentrations were independent predictors of PSA failure. CS was a stronger prognostic feature than tumor grade and could predict outcome for patients with moderately differentiated tumors. Patients with a low CS concentration had significantly better progression-free survival following radical prostatectomy than patients with high levels of CS (Kaplan-Meier plot, 91% versus 49% PSA progression free at 5 years, respectively, P = 0.0038). Only postoperative pathological indices (extracapsular extension, surgical margins) were stronger predictors than CS. We conclude that measurement of prostatic CS concentrations at diagnosis may allow stratification of patients with early-stage prostate cancer for adjunctive or alternate therapies.

Elevated levels of versican but not decorin predict disease progression in early-stage prostate cancer.

Patients with clinically localized prostate cancer who might be cured by aggressive management are not easily identified using current clinical information. Additional, more accurate, biomarkers of tumor behavior need to be identified to improve clinical outcome. Our previous studies indicated that the concentration of the glycosaminoglycan chondroitin sulfate in prostatic stroma might be a useful biomarker of disease progression in early-stage prostate cancer. In this study, two chondroitin sulfate proteoglycans, versican and decorin, were investigated. Versican and decorin were immunolocalized to the periacinar and peritumoral fibromuscular stroma in sections of nonmalignant and malignant human prostate tissues. Video image measurements indicated that the concentrations of both proteoglycans were increased in the prostatic tissue of men with early-stage prostate cancer compared with tissue from men without cancer (P = 0.0006). Cox's univariate analysis indicated that increases in versican concentration but not in that of decorin were associated with increased risk of prostate-specific antigen (PSA) progression. Versican concentration was compared with other clinical or biological features of prognosis in two-variable regression analyses. Versican and serum PSA concentrations were independent predictors of PSA progression. Versican was a stronger prognostic factor than tumor grade, and it could predict outcome for patients with moderately differentiated tumors. Patients with low versican concentration had significantly better progression-free survival than patients with high levels of versican (Kaplan-Meier plot, 89% versus 27% PSA progression-free at 5 years, respectively; P = 0.0001). We conclude that the measurement of prostatic concentrations of versican, a molecule with reported anticellular adhesive properties, may be a useful marker of disease progression in patients with early-stage prostate cancer and that further study of versican in other patient cohorts is warranted.

Purging of myeloma cells using all-trans retinoic acid in a mouse model.

OBJECTIVE: The 5T33 murine model of multiple myeloma was used to investigate the potential of all-trans retinoic acid (ATRA) to purge clonogenic myeloma cells from autologous hemopoietic stem-cell harvests by differentiating immature 5T33 cells into terminal-stage plasma cells with limited repopulation capacity. MATERIALS AND METHODS: 5T33 cells were treated with 10 microM ATRA and the effect on cell clonogenicity was determined by measuring the time to paraprotein detection in C57Bl/KaLwRij mice compared to control animals. Cell differentiation and apoptosis following ATRA treatment were investigated using flow cytometry and caspase-3 assay. Treatment with ATRA resulted in a 33% reduction in the in vitro cloning efficiency of 5T33 cells. Reduced in vitro clonogenicity of 5T33 cells following ATRA treatment was supported by a 16-49% increase in the time taken for C57Bl/KaLwRij mice to develop paraprotein following injection of 5T33 cells pretreated with ATRA for 8 days. Although ATRA was shown not to alter the in vitro growth characteristics of 5T33 cells, significant inhibition of apoptosis was observed. RESULTS: Treatment with ATRA also resulted in an increase in the proportion of 5T33 cells expressing the CD54 adhesion molecule, which is known to be highly expressed on mature myeloma cells. CONCLUSION: The ability of ATRA to decrease the clonogenicity of 5T33 cells in vitro and increase the time to disease development in vivo suggests that this drug may be useful for purging autologous stem cell harvests in the clinical setting.

Prolongation of sheep corneal allograft survival by ex vivo transfer of the gene encoding interleukin-10.

BACKGROUND: Modification of a donor cornea by gene therapy ex vivo has potential to modulate irreversible rejection, the major cause of corneal graft failure. Our aim was to transfer the gene encoding mammalian IL-10 to ovine donor corneas and to determine subsequent orthotopic corneal allograft survival in an outbred sheep model. METHODS: The replicative capacity of ovine corneal endothelium was determined by autoradiography after deliberate injury. A replication-defective adenovirus was used to deliver the lacZ reporter gene to ovine corneas and transfected corneas were organ-cultured in vitro to allow transfection efficiency, duration of reporter gene expression, and toxicity attributable to the vector to be determined. A cDNA encoding full-length ovine IL-10 was cloned into an adenoviral vector that was used to transfect donor corneas ex vivo before transplantation. Orthotopic penetrating corneal transplantation was performed in outbred sheep. RESULTS: Sheep corneal endothelium was found to be essentially amitotic. Transfection of > 70% corneal endothelial cells was achieved with the viral vector and expression was maintained for 28 days in vitro. IL-10 mRNA was detectable in transfected, organ-cultured corneas for 21 days in vitro. Donor corneas transfected with cDNA encoding IL-10 showed significantly prolonged survival after penetrating keratoplasty (median 55 days, range 19 > or =300 days) compared with control corneas (median 20.5 days, range 18-32 days, P=0.011). CONCLUSION: Local gene therapy-mediated expression of the immunomodulatory cytokine IL-10 has the potential to reduce the incidence of corneal graft rejection and to prolong corneal allograft survival.

Quantitation of targets for PCR by use of limiting dilution.

We describe a general method to quantitate the total number of initial targets present in a sample using limiting dilution, PCR and Poisson statistics. The DNA target for the PCR was the rearranged immunoglobulin heavy chain (IgH) gene derived from a leukemic clone that was quantitated against a background of excess rearranged IgH genes from normal lymphocytes. The PCR was optimized to provide an all-or-none end point at very low DNA target numbers. PCR amplification of the N-ras gene was used as an internal control to quantitate the number of potentially amplifiable genomes present in a sample and hence to measure the extent of DNA degradation. A two-stage PCR was necessary owing to competition between leukemic and non-leukemic templates. Study of eight leukemic samples showed that approximately two potentially amplifiable leukemic IgH targets could be detected in the presence of 160,000 competing non-leukemic genomes. The method presented quantitates the total number of initial DNA targets present in a sample, unlike most other quantitation methods that quantitate PCR products. It has wide application, because it is technically simple, does not require radioactivity, addresses the problem of excess competing targets and estimates the extent of DNA degradation in a sample.

Effects of oestradiol-17 beta and 5 alpha-dihydrotestosterone on guinea-pig prostate smooth muscle cell proliferation and steroid receptor expression in vitro.

Smooth muscle cells (SMCs) are the major cellular component of the prostatic stroma. The aim of this study was to examine the effects of oestradiol-17 beta (OE2) and 5 alpha-dihydrotestosterone (DHT) on the proliferation of guinea-pig prostate SMCs in vitro. OE2 stimulated SMC DNA synthesis at all concentrations examined. At a plating density of 3.0 x 10(4) cells/cm2, maximal incorporation of [3H]thymidine (136% of control) was observed after 36 h of treatment with 1 nmol OE2/l. At the same plating density, DHT had an inhibitory effect on SMC DNA synthesis, with maximal effects (73% of control) being observed 24 h after treatment with 1 nmol DHT/l. These effects of OE2 and DHT were prevented by co-incubation with specific steroid receptor antagonists. At a threefold lower plating density (1.0 x 10(4) cells/cm2), the maximal stimulatory and inhibitory effects of OE2 and DHT were delayed by approximately 24 and 12 h respectively. At the lower plating density, a biphasic effect of DHT was observed on DNA synthesis; DHT was both inhibitory and stimulatory. Maximal inhibition (71% of control) and maximal stimulation (168% of control) were observed after 36 and 134 h treatment with DHT respectively. At the lower plating density, longer term treatment of SMC cultures with OE2 and DHT also resulted in an increase in cell number. After 7 days of treatment with OE2 and DHT, cell number increased by 13% and 12% respectively. When OE2 and DHT were added in combination, the short-term inhibitory effect of DHT on SMC DNA synthesis was dominant over the stimulatory effect of OE2. Treatment with DHT for 24 h significantly inhibited OE2-induced stimulation of [3H]thymidine incorporation, irrespective of the prior duration of OE2 treatment. At the lower plating density, OE2 also decreased oestrogen receptor (ER) mRNA levels to 38% of control levels after 24 h of treatment. ER mRNA levels remained repressed until 72 h after treatment with OE2, and returned to control values following 96 h of treatment. Both the androgen-induced inhibition and stimulation of DNA synthesis observed following treatment of SMCs with 1 nmol DHT/l were associated with a reduction in androgen receptor (AR) mRNA levels. At an intermediate time (i.e. 48 h after commencement of treatment with DHT) AR mRNA levels were increased more than twofold over control levels.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of exposure to 900 MHz radiofrequency radiation on intrachromosomal recombination in pKZ1 mice.

Radiofrequency (RF) radiation emitted from mobile phones is not considered to be directly genotoxic, but it may have downstream effects on cellular DNA. We studied the effect of 4 W/kg pulsed 900 MHz RF radiation on somatic intrachromosomal recombination in the spleen in the pKZ1 recombination mutagenesis model. Somatic intrachromosomal recombination inversion events were detected in spleen tissue of pKZ1 mice by histochemical staining for E. coli beta-galactosidase protein in cells in which the lacZ transgene has undergone an inversion event. pKZ1 mice were exposed daily for 30 min to plane-wave fields of 900 MHz with a pulse repetition frequency of 217 Hz and a pulse width of 0.6 ms for 1, 5 or 25 days. Three days after the last exposure, spleen sections were screened for DNA inversion events. There was no significant difference between the control and treated groups in the 1- and 5-day exposure groups, but there was a significant reduction in inversions below the spontaneous frequency in the 25-day exposure group. This observation suggests that exposure to RF radiation can lead to a perturbation in recombination frequency which may have implications for recombination repair of DNA. The biological significance of a reduction below the spontaneous frequency is not known. The number of mice in each treatment group in this study was small (n = 10 or n = 20). Therefore, repetition of this study with a larger number of animals is required to confirm these observations.

Rapid method for detecting monoclonality in B cell lymphoma in lymph node aspirates using the polymerase chain reaction.

AIMS: To use the polymerase chain reaction to detect monoclonality at the immunoglobulin heavy chain gene locus in cells derived from lymph node aspirates. METHODS: A nested two-stage polymerase chain reaction (PCR) for the VDJ region of the immunoglobulin heavy chain gene was used to detect monoclonality. The total number of cells available for diagnosis by PCR in lymph node aspirates was between 10(4) and 10(5). RESULTS: A monoclonal band was detected in 21 of 25 malignant B-lymphomas. The other four specimens gave polyclonal bands. Specimens from reactive lymph nodes produced polyclonal bands in 14 cases, no product in two cases, and one specimen gave two monoclonal bands. Polyclonal bands were obtained for three Hodgkin's lymphoma samples and five metastatic carcinomas. Four metastatic carcinoma samples gave no amplification. CONCLUSIONS: Detection of monoclonality in a cell population is strongly suggestive of malignant disease. The simple PCR method presented here should complement conventional cytological and immunological methods for diagnosis of malignancy by lymph node aspirates.

Use of DNA polymorphisms and the polymerase chain reaction to examine the survival of a human limbal stem cell allograft.

PURPOSE: The extent to which limbal epithelial stem cell allografts will repopulate the human corneal ocular surface, and the time frame over which such cells survive, are uncertain. We investigated the survival of donor-derived epithelial cells after limbal stem cell allotransplantation in a patient with bilateral limbal stem cell failure by using short tandem-repeat DNA polymorphisms to distinguish donor and recipient cells. METHODS: Epithelial cells were harvested by impression cytology from the grafted eye before and at various times after transplantation. DNA was extracted and amplified by the polymerase chain reaction at an informative locus, D8S264. RESULTS: Cells of donor genotype were present over the grafted areas at the time of surgery but were not detected in the central cornea until 12 weeks postoperatively, indicating that repopulation of the epithelial surface from transplanted limbal stem cells took considerable time. However, by the 20th postoperative week, only recipient-type cells were detected in the grafted eye, despite systemic immunosuppression of the recipient with azathioprine and cyclosporine. CONCLUSIONS: Discrimination between donor and recipient cells on the ocular surface after limbal allotransplantation was possible using genotypic variation at DNA polymorphic sites (microsatellites). Long-term survival of donor cells after limbal transplantation did not occur in this patient. Detection of DNA polymorphisms amplified by the polymerase chain reaction is a simple, rapid, and noninvasive method of following the course of transplanted cells at the ocular surface.

Inversion due to intrachromosomal recombination produced by carcinogens in a transgenic mouse model.

Somatic intrachromosomal recombination (SICR) can result in inversions and deletions in the DNA. pKZ1 mice possess an Escherichia coli (E. coli) lacZ transgene which is only expressed after a DNA inversion involving the transgene occurs. The E. coli beta-galactosidase protein can then be detected in frozen tissue sections using a chromogenic substrate. Therefore, pKZ1 mice can be used to detect SICR inversion events in vivo in different tissues. We have tested the pKZ1 mouse for its potential as a general mutagenesis model for detecting SICR in spleen in response to carcinogens which have widely different mechanisms of genotoxicity. Animals were given a single exposure of carcinogen and spleen cells were examined 3 days later for inversion events by histochemical staining of tissue sections. Mitomycin C, X-irradiation, etoposide and methylene chloride caused significant induction of inversion events in spleen tissue, ranging from 1.6- to 4.2-fold induction with the doses used here. This is the first time that inversion events induced by these carcinogens have been specifically studied in vivo in a mouse model and the findings expand the repertoire of mutation events known to be caused by these agents. We suggest that the pKZ1 mouse can be used as a general mutagenesis model for detection of SICR events and is likely to be a useful model for studying the mechanism of SICR in response to DNA damaging agents.