Ten-year outcome of early childhood traumatic brain injury: Diffusion tensor imaging of the ventral striatum in relation to executive functioning.

PRIMARY OBJECTIVE: The long-term effects of TBI on verbal fluency and related structures, as well as the relation between cognition and structural integrity, were evaluated. It was hypothesized that the group with TBI would evidence poorer performance on cognitive measures and a decrease in structural integrity. RESEARCH DESIGN: Between a paediatric group with TBI and a group of typically-developing children, the long-term effects of traumatic brain injury were investigated in relation to both structural integrity and cognition. Common metrics for diffusion tensor imaging (DTI) were used as indicators of white matter integrity. METHODS AND PROCEDURES: Using DTI, this study examined ventral striatum (VS) integrity in 21 patients aged 10-18 years sustaining moderate-to-severe traumatic brain injury (TBI) 5-15 years earlier and 16 demographically comparable subjects. All participants completed Delis-Kaplan Executive Functioning System (D-KEFS) sub-tests. MAIN OUTCOMES AND RESULTS: The group with TBI exhibited lower fractional anisotropy (FA) and executive functioning performance and higher apparent diffusion coefficient (ADC). DTI metrics correlated with D-KEFS performance (right VS FA with Inhibition errors, right VS ADC with Letter Fluency, left VS FA and ADC with Category Switching). CONCLUSIONS: TBI affects VS integrity, even in a chronic phase, and may contribute to executive functioning deficits.

Sulforaphane induces phase II detoxication enzymes in mouse skin and prevents mutagenesis induced by a mustard gas analog.

Mustard gas, used in chemical warfare since 1917, is a mutagenic and carcinogenic agent that produces severe dermal lesions for which there are no effective therapeutics; it is currently seen as a potential terrorist threat to civilian populations. Sulforaphane, found in cruciferous vegetables, is known to induce enzymes that detoxify compounds such as the sulfur mustards that react through electrophilic intermediates. Here, we observe that a single topical treatment with sulforaphane induces mouse epidermal levels of the regulatory subunit of glutamate-cysteine ligase, the rate-limiting enzyme in glutathione biosynthesis, and also increases epidermal levels of reduced glutathione. Furthermore, a glutathione S-transferase, GSTA4, is also induced in mouse skin by sulforaphane. In an in vivo model in which mice are given a single mutagenic application of the sulfur mustard analog 2-(chloroethyl) ethyl sulfide (CEES), we now show that therapeutic treatment with sulforaphane abolishes the CEES-induced increase in mutation frequency in the skin, measured four days after exposure. Sulforaphane, a natural product currently in clinical trials, shows promise as an effective therapeutic against mustard gas.

Molecular cloning and characterization of a novel mouse epidermal differentiation gene and its promoter.

The transcription factor E2F1 is an important regulator of cell proliferation, apoptosis, and differentiation. A novel mouse gene (Eig3) was originally identified as up-regulated in E2F1-overexpressing keratinocytes by the rapid analysis of gene expression technique. An apparently full-length cDNA and a 2.8-kb genomic fragment containing the entire gene have been cloned. Sequence comparisons suggest that Eig3 is homologous to a human epidermal differentiation gene, XP5, and belongs to a family of at least 10 murine genes that are related to the small proline-rich genes involved in skin differentiation. Eig3 was expressed in adult mouse stomach and epidermis and overexpressed in keratinocytes transgenic for E2F1 or E2F4, but not in c-myc transgenics. Interestingly, Eig3 expression was highly increased in mouse skin papillomas but not in squamous carcinomas. Since there is no E2F consensus binding sequence in the promoter or first intron of Eig3, E2F regulation may be indirect.

Impact of laparoscopic cholecystectomy: a population-based study.

BACKGROUND: We assessed the effect of the introduction of laparoscopic cholecystectomy on surgical outcomes in routine practice. METHODS: Hospital discharge and death-certificate data were linked for all patients undergoing cholecystectomy (n=85120) in Scottish public-sector hospitals (n=51) between January, 1981, and June, 1999. The primary endpoints were cholecystectomy rate, hospital stay, and postoperative mortality. Regression methods were used to examine the effect of laparoscopic experience and surgeon caseload on postoperative mortality and hospital stay. FINDINGS: From 1989 to 1999, the proportion of cholecystectomies done laparoscopically rose from none to 80%, and the age-standardised cholecystectomy rate increased by 20% (95% CI 15-26). Postoperative mortality did not change in the 1990s (odds ratio 0.99 [0.7-1.4], p=0.99). The mean postoperative hospital stay fell from 8.0 (SD 3.7) to 2.9 (3.2) days. There was wide variation between hospitals in the proportion of cholecystectomies done laparoscopically and in average hospital stay. For individual surgeons, increasing laparoscopic experience and annual caseload were associated with higher proportions of laparoscopic procedures and shorter hospital stays. Postoperative mortality was higher during the first ten laparoscopic cholecystectomies done by a surgeon (compared with >200 procedures, odds ratio 2.3 [1.2-4.6], p=0.015). INTERPRETATION: The laparoscopic method reduced hospital stay but had no overall effect on postoperative mortality. Studies to assess the appropriateness of the increased cholecystectomy rate are merited. The wide variation in the proportion done laparoscopically, together with evidence of better results for surgeons doing more procedures, suggests scope for further reductions in hospital stay and morbidity.

Effects of estrogen on global gene expression: identification of novel targets of estrogen action.

The important role played by the sex hormone estrogen in disease and physiological processes has been well documented. However, the mechanisms by which this hormone elicits many of its normal as well as pathological effects are unclear. To identify both known and unknown genes that are regulated by or associated with estrogen action, we performed serial analysis of gene expression on estrogen-responsive breast cancer cells after exposure to this hormone. We examined approximately 190,000 mRNA transcripts and monitored the expression behavior of 12,550 genes. Expression levels for the vast majority of those transcripts were observed to remain constant upon 17beta estradiol (E2) treatment. Only approximately 0.4% of the genes showed an increase in expression of > or =3-fold by 3 h post-E2 treatment. We cloned five novel genes (E2IG1-5), which were observed up-regulated by the hormonal treatment. Of these the most highly induced transcript, E2IG1, appears to be a novel member of the family of small heat shock proteins. The E2IG4 gene is a new member of the large family of leucine-rich repeat-containing proteins. On the basis of architectural and domain homology, this gene appears to be a good candidate for secretion in the extracellular environment and, therefore, may play a role in breast tissue remodeling and/or epithelium-stroma interactions. Several interesting genes with a potential role in the regulation of cell cycle progression were also identified to increase in expression, including Pescadillo and chaperonin CCT2. Two putative paracrine/autocrine factors of potential importance in the regulation of the growth of breast cancer cells were identified to be highly up-regulated by E2: stanniocalcin 2, a calcium/phosphate homeostatic hormone; and inhibin-beta B, a TGF-beta-like factor. Interestingly, we also determined that E2IG1 and stanniocalcin 2 were exclusively overexpressed in estrogen-receptor-positive breast cancer lines, and thus they have the potential to serve as breast cancer biomarkers. This data provides a comprehensive view of the changes induced by E2 on the transcriptional program of human E2-responsive cells, and it also identifies novel and previously unsuspected gene targets whose expression is affected by this hormone.

A novel approach for analyzing the structure of DNA modified by Benzo[a]pyrene diol epoxide at single-molecule resolution.

Benzo[a]pyrene diol epoxide (BPDE) has been shown to bind specifically to the exocyclic amino group of deoxyguanosine in duplex DNA. Interestingly, this metabolite exhibits stereoselectivity in its tumorigenic and mutagenic effects. It is thought that local DNA conformation is altered at the site of the adduct, resulting in aberrant biological processes, and that in certain sequence contexts BPDE-DNA adducts induce bends in the DNA. In the work presented here, we compared DNA structural alterations of BPDE-modified DNA and unmodified DNA via tapping mode atomic force microscopy (AFM). DNA fragments 366 base pairs (bp) in length were generated by PCR from the duplicated multiple-cloning site of pBEND2 inserted into pGEM-3Zf(-), and either mock-modified or treated with BPDE to give modification levels between 1 and 5% of the nucleotides. Control or BPDE-modified DNA was adsorbed to mica and visualized in air by AFM. The contour lengths and end-to-end lengths of individual molecules were measured. The ratio of end-to-end distance to contour length was significantly smaller for modified DNA molecules than for the unmodified DNA preparation, although the frequency distributions of the contour lengths were similar for the two preparations. This suggests BPDE-DNA adducts cause significant bending of DNA molecules, confirming previous conclusions based on more indirect measurements. The average induced bend angle for BPDE-DNA adducts is estimated to be at least 30 degrees.

Regulation of BRCA1 expression by the Rb-E2F pathway.

Inheritance of a mutant allele of the breast cancer susceptibility gene BRCA1 confers increased risk of developing breast and ovarian cancers. Likewise, inheritance of a mutant allele of the retinoblastoma susceptibility gene (RB1) results in the development of retinoblastoma and/or osteosarcoma, and both alleles are often mutated or inactivated in sporadic forms of these and other cancers. We now demonstrate that the product of the RB1 gene, Rb, regulates the expression of the murine Brca1 and human BRCA1 genes through its ability to modulate E2F transcriptional activity. The Brca1 gene is identified as an in vivo target of E2F1 in a transgenic mouse model. The Brca1 promoter contains E2F DNA-binding sites that mediate transcriptional activation by E2F1 and repression by Rb. Moreover, ectopic expression of cyclin D1 and Cdk4 can stimulate the Brca1 promoter in an E2F-dependent manner, and this is inhibited by coexpression of the p16(INK4a) cyclin-dependent kinase inhibitor. The human BRCA1 promoter also contains a conserved E2F site and is similarly regulated by E2F1 and Rb. This functional link between the BRCA1 and Rb tumor suppressors may provide insight into the mechanism by which BRCA1 inactivation contributes to cancer development.

Rapid analysis of gene expression (RAGE) facilitates universal expression profiling.

Current techniques for analysis of gene expression either monitor one gene at a time, for example northern hybridization or RT-PCR methods, or are designed for the simultaneous analysis of thousands of genes, for example microarray hybridization or serial analysis of gene expression. To provide a flexible, intermediate scale alternative, a PCR-based method for the rapid analysis of gene expression has been developed which allows expression changes to be determined in either a directed search of known genes, or an undirected survey of unknown genes. A single set of reagents and reaction conditions allows analyses of most genes in any eukaryote. The method is useful for assaying on the order of tens to hundreds of genes in multiple samples. Control experiments indicate reliable detection of changes in gene expression 2-fold and greater, and sensitivity of detection better than 1 in 10 000. Analyses of over 400 genes in a mouse system transgenic for the E2F1 gene have identified several new downstream targets of E2F1, including Brca1 and Cdk7, in addition to several unidentified genes that are upregulated in the transgenic mice. Changes in expression of several genes related to apoptosis suggest a possible potentiation of apoptotic pathways in the transgenic keratinocytes.

Geographic, demographic, and socioeconomic variations in the investigation and management of coronary heart disease in Scotland.

OBJECTIVE: To determine whether age, sex, level of deprivation, and area of residence affect the likelihood of investigation and treatment of patients with coronary heart disease. DESIGN, PATIENTS, AND INTERVENTIONS: Routine discharge data were used to identify patients admitted with acute myocardial infarction (AMI) between 1991 and 1993 inclusive. Record linkage provided the proportion undergoing angiography, percutaneous transluminal coronary angioplasty (PTCA), and coronary artery bypass grafting (CABG) over the following two years. Multiple logistic regression analysis was used to determine whether age, sex, deprivation, and area of residence were independently associated with progression to investigation and revascularisation. SETTING: Mainland Scotland 1991 to 1995 inclusive. MAIN OUTCOME MEASURES: Two year incidence of angiography, PTCA, and CABG. Results-36 838 patients were admitted with AMI. 4831 (13%) underwent angiography, 587 (2%) PTCA, and 1825 (5%) CABG. Women were significantly less likely to undergo angiography (p < 0.001) and CABG (p < 0.001) but more likely to undergo PTCA (p < 0.05). Older patients were less likely to undergo all three procedures (p < 0.001). Socioeconomic deprivation was associated with a reduced likelihood of both angiography and CABG (p < 0.001). There were significant geographic variations in all three modalities (p < 0.001). CONCLUSION: Variations in investigation and management were demonstrated by age, sex, geography, and socioeconomic deprivation. These are unlikely to be accounted for by differences in need; differences in clinical practice are, therefore, likely.

Enhancing the power of record linkage involving low quality personal identifiers: use of the best link principle and cause of death prior likelihoods.

The Heartstart Scotland study collects details of all resuscitation attempts carried out by the Scottish Ambulance Service. The linkage between records for Heartstart study subjects who died before admission to a hospital and the national file of death records maintained by the Registrar General for Scotland is described. The conditions under which the Heartstart data is collected make it inevitable that the personal identifying information on which linkage must rely tends to be relatively incomplete and of low accuracy. The linkage process was able to use the best-link principle to take maximum advantage of the fact that, because the Heartstart subjects involved had died, there was an extremely high a priori probability that they would be represented on the national deaths file. In addition, although no cause of death information was recorded on the Heartstart records, a priori expectations of the distribution of causes of death among linked death records were used. Despite these enhancements, however, clerical resolution of a proportion of the potential links generated by the automatic algorithm significantly improved the accuracy of the linkage.

Mixed effects of 2,6-dithiopurine against cyclophosphamide mediated bladder and lung toxicity in mice.

2,6-Dithiopurine (DTP) has been proposed as a possible chemopreventive agent because of its ability to react with electrophiles. Acrolein, an electrophilic metabolite of cyclophosphamide (CP) involved in the toxicities of this anticancer drug, can be scavenged by DTP. The present study examined the effect of DTP treatment on CP-mediated bladder and lung toxicity in male ICR mice. Mice fed a diet containing 4% DTP that were treated intraperitoneally (i.p.) with 350 mg/kg CP showed no significant bladder damage (measured as bladder blood content at 48 h) with respect to the group fed a control diet. DTP (50 and 100 mg/kg), given i.p. 0.5 and 7 h after the initial injection of CP, also prevented the bladder damage when compared with the group receiving CP alone. Surprisingly, although neither parenteral CP nor DTP alone caused any mortality at these doses, the combined treatment resulted in 67% mortality within 3 days. At 24 h after CP + DTP, blood urea nitrogen was elevated 6-fold and urine volumes decreased by 70%. Histopathological analyses revealed a diffuse myocardial degeneration and necrosis, severe granular degeneration in the liver, abundant cellularity and infiltrates in interalveolar spaces in the lung and swollen nephron epithelial cells with some necrosis. All mice survived treatment when the dose of CP was lowered to 250 and 25-75 mg/kg DTP was given i.p. 0.5 and 7 h after CP. These DTP regimens reduced the degree of CP-induced lung toxicity, measured by [3H]thymidine incorporation into lung DNA 7 days after CP, in a dose-dependent manner. DTP (75 mg/kg) also reduced CP-induced lung fibrosis estimated by lung hydroxyproline content 28 days after CP. Analyses of urine from mice given CP + DTP revealed large amounts of the metabolic product dithiouric acid, smaller amounts of the parent DTP and several smaller peaks. The major unique metabolite peak was collected and analyzed by mass spectrometry, but did not correspond to either acrolein-DTP or acrolein-dithiouric acid. Thus, either very small amounts of an acrolein adduct are generated, the adduct is broken down to an unidentified product, or the ability of DTP to prevent CP-induced lung and bladder damage is related to some other mechanism. The possibility that mercapturic acid metabolites of acrolein released the parent electrophile in the urine was not supported by the finding that probenecid did not prevent CP-induced bladder toxicity.

High-affinity binding of the cell cycle-regulated transcription factors E2F1 and E2F4 to benzo[a]pyrene diol epoxide-DNA adducts.

Previous studies indicated that DNA adducts formed by a carcinogenic diol epoxide, 7r,8t-dihydroxy-9t, 10t-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE), can increase the affinity of the transcription factor Sp1 for DNA sequences that are not normally specific binding sites. It was suggested that adduct-induced bends in the DNA were responsible for this behavior. The cell cycle-regulated transcription factor E2F is also known to bend DNA upon binding. When partially purified E2F was tested in a gel mobility-shift assay, binding to a target DNA containing two consensus E2F-binding sites was enhanced by prior modification of the DNA with BPDE. Recombinant human E2F1, E2F4, and DP1 fusion proteins were affinity purified from bacteria expressing these genes. A combination of either E2F1 or E2F4 with their dimerization partner, DP1, gave preparations that exhibited binding to the E2F site-containing DNA fragment. In both cases, the proteins exhibited much higher apparent affinity for BPDE-modified DNA than for unmodified DNA. In addition, BPDE-modified DNA was a better competitor for the binding than unmodified DNA. Heterologous DNA that contained no consensus E2F binding motifs also competed well for E2F binding when modified with BPDE. In contrast, transcription factor that does not bend DNA appreciably (GAL4) did not show enhanced affinity for BPDE-modified DNA. These findings suggest that numerous transcription factors that bend DNA may bind with anomalously high affinity to sequences that contain carcinogen-DNA adducts.

Induction of mammary cancer and lymphoma by multiple, low oral doses of 7,12-dimethylbenz[a]anthracene in SENCAR mice.

Existing models of mouse mammary carcinogenesis induced by the model polycyclic aromatic hydrocarbon 7,12-dimethylbenz[a]anthracene (DMBA) typically use a small number of bolus doses applied intragastrically. In contrast to this, typical human exposures to carcinogens are thought to be at lower doses and to occur with chronic or sporadic timing. When the classical dosage (1 mg DMBA given once a week for 6 weeks) was split into five daily doses of 200 microg given intragastrically to female SENCAR mice each week for 6 weeks, toxicity was high and the major tumor type seen was lymphoma. Lowering the dose to 60 microg/day gave less toxicity, a 75% incidence of lymphoma and a 30% incidence of mammary carcinoma. However, 20 microg DMBA given five times per week for 6 weeks resulted in a 65-70% incidence of mammary carcinoma within approximately 50 weeks. This represents a 50-fold lower daily dosage of DMBA than that used in the classical model. DNA was prepared from 10 mammary adenocarcinomas and 10 lymphomas and exons 1 and 2 of the H-ras1, K-ras and N-ras genes were sequenced using PCR techniques. Mutations altering codons 12 or 61 of one of the ras family genes were found in 4/10 mammary carcinomas and 5/10 lymphomas. Three mammary tumors exhibited codon 61 mutations, one in each of the genes studied, and a fourth tumor contained a codon 12 mutation in the K-ras gene. Among the lymphomas, two mutations in codon 12 of K-ras, one mutation in codon 61 of K-ras and two mutations in codon 61 of N-ras were also found. Each of the mutations could be interpreted as a G-->T or A-->T transversion. It is suggested that the high incidence of lymphoma at the higher, repetitive doses may be related to immunotoxicity. These low dose models of lymphomagenesis and mammary carcinogenesis should prove useful for tests of chemopreventive agents that target the initiation phase of carcinogenesis.

Disruption of transcription in vitro and gene expression in vivo by DNA adducts derived from a benzo[a]pyrene diol epoxide located in heterologous sequences.

Previous studies indicated a high affinity of the transcription factor Sp1 for DNA adducts derived from benzo[a]pyrene diol epoxide (BPDE) in sequences that are not normal binding sites for Sp1. We tested for functional effects of this phenomenon in three systems in which transcription is Sp1-dependent. In an in vitro, Sp1-dependent transcription system addition of heterologous plasmid DNA containing BPDE adducts abolished production of a specific run-off transcript. This inhibition was not seen with unmodified plasmid DNA, and could be overcome by addition of purified Sp1 protein. In SL2 insect cells, high-level expression of an Sp1-dependent reporter gene, which was dependent on co-transfection of an Sp1 expression vector, was inhibited >95% by co-transfection of heterologous DNA containing BPDE adducts. This inhibition could be partially overcome by increasing the amount of the Sp1 expression vector in the transfections. In human C33A cells, expression of a transfected reporter gene driven by a GC box containing fragment of the human E2F1 promoter was enhanced by co-transfection of an Sp1 expression plasmid. Expression was inhibited 3-6-fold by co-transfection of heterologous DNA containing BPDE-DNA adducts. A similar inhibition was seen in human SAOS-2 cells, which lack functional p53 protein. These data are consistent with functionally significant sequestration of the Sp1 transcription factor by BPDE-DNA adducts in all three systems. Altered availability of transcription factors such as Sp1 in carcinogen-treated cells may disrupt patterns of gene expression.

DNA sequence and nucleosome placement on the murine fibroblast growth factor-4 gene.

A large portion of the 5' flanking region of the fibroblast growth factor-4 (FGF-4) gene has been sequenced, but only 38 bp of sequence information past the end of the last exon of this gene has been described. In this study, a total of 2769 bp of nucleotide sequence down-stream of the last exon of FGF-4 (GenBank #U43515) was determined by a combination of deletional subcloning and primer walking. In addition, we have used the theoretical algorithm of Calladine and Drew to predict the rotational positioning of nucleosomes throughout the entire FGF-4 gene.

Kinetics of the reaction of a potential chemopreventive agent, 2,6-dithiopurine, and its major metabolite, 2,6-dithiouric acid, with multiple classes of electrophilic toxicants.

Purinethiols are a class of potential cancer chemopreventive agents that exhibit nucleophilic scavenging activity against the carcinogenic electrophile benzo[a]pyrene diol epoxide (BPDE). Of the purinethiols tested previously, 2,6-dithiopurine (DTP), exhibited the highest scavenging activity for BPDE when tested either in vitro or in vivo. Sulfur-based nucleophiles are typically classified as "soft" nucleophiles, showing selectivity in nucleophilic substitution reactions for "soft", easily polarizable electrophiles. It was of interest to determine whether electrophilic toxicants other than BPDE react facilely with DTP, and whether 2,6-dithiouric acid (DUA), the major in vivo metabolite of DTP, also has scavenging activity. Four diverse toxicants tested in the present work, acrolein, melphalan, dimethyl sulfate, and cisplatin, all react facilely with DTP in vitro near neutral pH. These toxicants are expected to react as "soft" electrophiles. Furthermore, each of these compounds, as well as BPDE, reacts with DUA with rate constants comparable to the analogous rate constants for reaction with DTP. In contrast, several toxicants classified as "hard" electrophiles (ethyl methanesulfonate, methylnitrosourea, ethylnitrosourea, 1-methyl-3-nitro-1-nitrosoguanidine) show no appreciable reaction with DTP. These results suggest that both DTP and its major metabolite act as "soft" nucleophiles in nucleophilic substitution reactions and may be effective in scavenging a wide range of toxicants that react as "soft" electrophiles.

The probability of occurrence of oligomer motifs in the human genome and genomic microheterogeneity.

A previously published method for predicting the frequency of random occurrence of a completely specified DNA oligomer in a longer sequence dataset has been generalized to allow degeneracy in the oligomer sequence. With this enhancement, several datasets consisting of sequences from the human genome were searched for the occurrence of consensus binding sites for a set of 13 transcription factors. Although because of the biological significance of these sequences one might predict that they would occur more often than the random frequency, many of the consensus oligomers were found at lower than expected frequencies. Several (G+C)-rich oligomers were found to be moderately over-represented, but this could be accounted for, in part, by the occurrence of (G+C)-rich tracts in the human sequences. Regions very high in (G+C) were found to occur at much higher frequencies than expected in the human genome, and this severely limits the usefulness of this approach for predicting the frequency of (G+C)-rich oligomers. Unexpectedly, more than 1% of the human genome consists of tracts at least 28 bp in length with a (G+C) content greater than 85%.

Interference of benzo[a]pyrene diol epoxide-deoxyguanosine adducts in a GC box with binding of the transcription factor Sp1.

Previous studies indicated that DNA adducts formed by the carcinogenic diol epoxide 7r,8t-dihydroxy-9t,10t-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) can increase the affinity of the transcription factor Sp1 for DNA sequences that are not normally specific binding sites. Whether adducts that form in the normal binding site, the GC box sequence, increase the affinity of Sp1 for the modified GC-box was not determined. Starting with a 23-nt sequence that contains two natural GC box sequences, site-specifically modified oligonucleotides were prepared with a single(+)-BPDE-deoxyguanosine adduct at one of three positions: the center of each GC-box or in between the two boxes. Four modified oligonucleotides were studied, two derived from cis addition of BPDE to the exocyclic amino group and two from trans addition. For three of these site-specifically modified oligonucleotides, there was a diminution in Sp1 affinity, whereas Sp1 binding to the fourth modified oligonucleotide was abolished. Furthermore, random modification of the oligonucleotide to a level of about 1 BPDE adduct per fragment slightly decreased the affinity for Sp1, and no evidence was found for a subpopulation of molecules with high affinity. These findings suggest that BPDE modification of the GC box does not lead to an increased affinity for Sp1. This is consistent with a model in which a BPDE-induced bend in the DNA mimics the conformation of the normal GC box:Sp1 complex, leading to high-affinity binding of Sp1 to non-Gc box sites.

A possible role in chemical carcinogenesis for epigenetic, heritable changes in gene expression.

Although genetic changes are clearly important in the initiation of carcinogenesis, there is reason to think that epigenetic changes may also play a role in the process. A key feature of carcinogenesis is the long latency between exposure to carcinogenic insults and the appearance of malignancy. Thus, if epigenetic changes are to be involved, they must somehow be inherited at each cell division without the continued presence of the carcinogen. I propose that self-perpetuating changes in patterns of gene expression are a plausible mechanism for an epigenetic component of carcinogenesis. Networks of transcription factors that regulate each other's and their own expression are known to control important developmental processes, particularly the determination of entire cell lineages. An inherent property of many such autoregulatory networks is the existence of two very distinct, stable steady-states, defined in terms of the concentration of each transcription factor in the network. In this report, I present a model in which an acute carcinogen exposure is postulated to shift such a network from one steady-state to the other, effectively turning on or off the expression of at least one of the genes. Because of the autoregulatory nature of the network, this new steady-state is stably inherited at each cell division. Such changes in gene expression may ultimately contribute to the malignant phenotype if the regulatory network affects genes important in cell-cycle checkpoints, maintenance of genome stability, signal transduction, or other processes that are altered in tumor cells.

Interaction of bulky chemical carcinogens with DNA in chromatin.

DNA damage is an important effect of treatment of cells and organisms with chemical carcinogens. Although much has been learned from in vitro studies of the reaction of carcinogens with purified DNA, in vivo the DNA is associated with a variety of histone and non-histone proteins in a complex and dynamic structure known as chromatin. The covalent interactions of bulky chemical carcinogens with chromatin are reviewed. Differences from bulk genomic DNA in adduct density are found in replicating, transcribing and nuclear matrix-bound DNA regions, and between DNA in nucleosome cores and linker DNA regions. These differences range from 2- to 3-fold for linker versus core, to approximately 8-fold close to a replication fork. Much remains to be done to determine the influences of non-histone proteins and higher order chromatin structure on carcinogen binding.