The PKZ1 recombination mutation assay: a sensitive assay for low dose studies.

The majority of mutation studies are performed at high doses of DNA damaging agents due to the insensitivity of most mutation assays. Extrapolation using a linear no-threshold (LNT) dose response model is then used to estimate the extent of possible DNA damage at lower doses. There is increasing evidence to suggest that the LNT model may not be correct at low doses of at least some DNA damaging agents. The pKZ1 in vivo and in vitro recombination assays have proven to be very sensitive for detection of changes in chromosomal inversion in lymphoid tissue in response to low doses of DNA damaging agents. Non-linear dose response curves for chromosomal inversion as an end-point have been identified at low doses of DNA damaging agents using this assay. Here, we review the inversion results obtained to date with the pKZ1 assays and discuss their suitability for low dose studies.

Frequency and genetic basis of MHC, beta-2-microglobulin and MEMO-1 loss of heterozygosity in sporadic breast cancer.

The Human Leukocyte Antigen (HLA) class I molecules are critical factors in T cell recognition of abnormal, including neoplastic, cells. Loss of HLA class I expression phenotypes, as defined by immunohistochemistry-based tests, have been previously described in many types of cancer. Here we describe a microsatellite marker DNA-based loss of heterozygosity (LOH) analysis of three distinct chromosomal regions which have been implicated in HLA class I expression on a cohort of 99 unselected sporadic breast cancer samples. These regions comprise the 4Mb major histocompatibility complex (MHC) region on chromosome 6p, which contains the HLA class I heavy chain loci and other genes responsible for antigen processing, the HLA class I light chain (beta-2-microglobulin, beta2m) gene on chromosome 15q, and the putative HLA class I modifier of methylation gene (MEMO-1) on chromosome 1p. Additional chromosome 6 markers were also employed to determine the likely genetic mechanism for MHC loss. We show that 25/99 (25%) of samples show allelic loss within the MHC, 28/95 informative samples (29%) show allelic loss of beta2m and 21/76 informative samples (28%) show allelic loss of MEMO-1. Approximately half of the samples are predicted to have compromised HLA class I gene expression due to LOH at one and/or other of these three loci. Sequencing of the remaining beta2m allele in samples displaying beta2m LOH failed to detect any additional intragenic mutations. Analysis of the frequency of samples showing LOH at either 0, 1, 2 or 3 of the genomic regions analyzed suggested clustering of tumors into either 'no loci loss' or '3 loci loss' categories. These results reveal major underlying genetic causes for the high level of HLA class I expression loss seen in breast cancer.

Lack of an effect of vitamin E on lifespan of mice.

It has been speculated that ageing results from accumulation of damage to macromolecules, particularly DNA, owing to the action of oxidising free radicals. This possibility would predict that administration of anti-oxidants might prolong lifespan, but previous data on this prediction are conflicting. Three groups of mice were exposed throughout life, from the time of conception until death, to 20, 40 and 400 mg/kg of vitamin E in the diet. No effect on lifespan was observed and the median lifespans in the three groups were 804, 830 and 801 days, respectively. The design of the study also enabled an effect of parental age on lifespan of female progeny to be sought, but no effect was detected.

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.

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.

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.

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.

The contribution of exogenous and endogenous mutagens to in vivo mutations.

There is abundant evidence of the potential for exogenous agents to cause cancer but the proportion of human cancers attributable to defined external agents is uncertain. With rare exceptions it is difficult to demonstrate a role for exogenous agents in increasing mutation above background rates. There are many sources of endogenous mutation including physico-chemical processes, free radicals and enzymatic processes controlling DNA damage and repair. Evidence for the role of diet and genetic factors as major determinants of endogenous mutagenesis is reviewed with reference to the spontaneous spectrum of mutations in human cells and the quantitative measurement of mutation frequency in dietary restriction and the senescence-accelerated mouse.

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.

Rapid detection of the factor V Leiden (1691 G > A) and haemochromatosis (845 G > A) mutation by fluorescence resonance energy transfer (FRET) and real time PCR.

A rapid method based on fluorescence resonance energy transfer (FRET) and real time polymerase chain reaction (PCR) was used to identify the haemochromatosis genotype in 112 individuals and the factor V genotype in 134 individuals. The results were compared with conventional methods based on restriction enzyme digestion of PCR products. The two methods agreed in 244 of the 246 individuals; for the other two individuals, sequencing showed that they had been incorrectly genotyped by the standard method but correctly genotyped by FRET. The simplicity, speed, and accuracy of real time PCR analysis using FRET probes make it the method of choice in the clinical laboratory for genotyping the haemochromatosis and factor V genes.

Minimal residual disease in childhood acute lymphoblastic leukaemia quantified by aspirate and trephine: is the disease multifocal?

The level of minimal residual disease (MRD) in marrow early in treatment strongly predicts outcome in childhood acute lymphoblastic leukaemia (ALL). Using PCR we studied 30 pairs of aspirates and trephines taken during induction treatment. Consensus PCR primers showed a monoclonal gene rearrangement in eight pairs, polyclonal rearrangement in 18 pairs and a monoclonal rearrangement only in the trephine in four pairs. MRD was quantified by leukaemia-specific primers in 22 pairs. There was a linear relationship between the logarithms of MRD levels of aspirate and trephine, with a residual variance which increased as the level of MRD fell. The mean level of MRD in the trephines was 4.1-fold greater than that in the aspirates, probably due to greater dilution of the aspirates with peripheral blood. The high variance at low levels of MRD could not be explained by measurement variation, which had an MRD-independent value of 0.42 log10 units, and was attributed to sampling variation due to patchiness of disease at low MRD levels. The magnitude of the variation was such that predictions of outcome could well be confounded for many patients. We suggest that MRD sampling variability could be minimized either by taking multiple marrow samples or by measuring MRD in peripheral blood.

A method for assessing strand breaks in DNA.

A simple method has been developed to assess strand breaks in extracted DNA. The method uses the enzyme terminal deoxynucleotidyl transferase (TDT) to incorporate labeled deoxycytidine triphosphate (dCTP) in the presence of dideoxy-CTP (ddCTP) which is added to ensure that the reaction goes to completion. Following development of the method, the extent of DNA degradation in 21 blood or bone marrow samples, which had varying degrees of DNA degradation, was measured by the TDT assay, by gel electrophoresis, or by a laborious PCR-based method which quantifies the number of amplifiable N-ras targets in a sample. The TDT assay was more sensitive at detecting strand breaks than electrophoresis and there was good correlation between the results of the TDT assay and the N-ras assay. The TDT assay was also used to demonstrate the development of strand breaks during induced apoptosis. The TDT assay is thus a simple and semiquantitative method to study strand breaks produced by DNA damage.

Induction of somatic intrachromosomal recombination inversion events by cyclophosphamide in a transgenic mouse model.

Somatic intrachromosomal recombination (SICR) can result in chromosomal inversion and deletion, mechanisms which are important in carcinogenesis. We have utilised a transgenic mouse model to study SICR inversion events in spleen cells. The transgenic construct is designed so that expression of an Escherichia coli lacZ transgene only occurs in a cell when an SICR inversion event occurs in the region of the transgene. The inversion events can then be detected by histochemical staining of frozen spleen sections for transgene expression and by polymerase chain reaction across the inversion breakpoints. The spontaneous inversion frequency in spleen rose 2-fold from 1.54 +/- 0.24 x 10(-4) (mean +/- SE) in 4-month-old transgenic mice to 3.12 +/- 0.67 x 10(-4) in 22-month-old mice. Four- or 8-month-old mice were treated with a single intraperitoneal injection of cyclophosphamide, with doses ranging from 0.01 to 100 mg/kg. The animals were killed 3 days after treatment. A significant induction of SICR inversions was detected at all doses with a 3.2-fold maximum induction of inversions detected at 10 mg/kg. These results suggest that the transgenic mouse model used here may be a sensitive model for studying the role of SICR in mutation and in studying risk assessment of environmental DNA-damaging agents.

Monitoring minimal residual disease in peripheral blood in B-lineage acute lymphoblastic leukaemia.

The use of peripheral blood rather than marrow has potential advantages for monitoring minimal residual disease during the treatment of leukaemia. To determine the feasibility of using blood, we used a sensitive polymerase chain reaction method to quantify leukaemia in the blood and marrow in 35 paired samples from 15 children during induction treatment. Leukaemic cells in the blood ranged from 1.1 x 10(-2) to < 9.4 x 10(-7) leukaemic cells/total cells, corresponding to 1.3 x 10(7) to < 2 x 10(3) leukaemic cells/l. In 15 paired samples, leukaemia could be quantified in both tissues and in 20 paired samples, leukaemia was not detected in one or both tissues so that only upper level limits could be set. In the former 15 pairs, the level of leukaemia in peripheral blood was directly proportional to that in marrow but was a mean of 11.7-fold lower. Leukaemia in blood was detected in 10/12 pairs in which the level in marrow was > 10(-4), but in only two of 13 pairs in which the level in marrow was < 10(-5). Patients studied at multiple time-points showed parallel declines in the number of leukaemic cells in both tissues. The results showed that leukaemia could be monitored in peripheral blood during induction therapy, and quantitative considerations based on the results suggest that monitoring of blood during post-induction therapy may be of value in detecting molecular relapse.