Ex vivo expanded telomerase-specific T cells are effective in an orthotopic mouse model for pancreatic adenocarcinoma

Telomerase activity is over-expressed in nearly all pancreatic carcinomas, but not in chronic pancreatitis. Here, we investigated various protocols for expansion of telomerase-specific T cells for adoptive cell transfer and their use in a syngeneic pancreatic carcinoma mouse model. Telomerase-specific T cells were generated by stimulation of splenocytes from peptide-immunized donor mice with either interleukin (IL)-2, IL-15, artificial antigen-presenting cells, anti-signalling lymphocyte activation molecule (SLAM) microbeads or allogeneic dendritic cells in combination with a limited dilution assay. T cells were tested for antigen specificity in vitro and for anti-tumour activity in syngeneic mice with orthotopically implanted tumours pretreated with cyclophosphamide. The immune cells from recipients were immunophenotyped. During a period of 2 weeks, the expansion approach using IL-2 was very successful in generating a high number of telomerase-specific CD8⁺ T cells without losing their function after adoptive cell transfer. Significantly slower tumour growth rate and less metastasis were observed after adoptively transferring telomerase specific CD8⁺ T cells, expanded using IL-2. Further investigations showed that anti-tumour efficacy was associated with a significant shift from naive CD8⁺ T cells to CD8⁺ central memory T cells, as well as recruitment of a high number of dendritic cells. Remarkable amounts of telomerase-specific T cells were detectable in the tumour. Generation of telomerase-specific T cells is feasible, whereat IL-2-based protocols seemed to be most effective and efficient. Antigen-specific T cells showed significant cytotoxic activity in a syngeneic, orthotopic mouse model, whereas central memory T cells but not effector memory T cells appear to be of high importance.     

Hyaluronidase expression induces prostate tumor metastasis in an orthotopic mouse model

Molecular mechanisms of prostate cancer progression are frequently studied in mice by orthotopic injection of aggressive cell lines, which yield primary tumors that spontaneously metastasize to lymph nodes. In this report, we characterized the human prostate carcinoma cell line 22Rv1 in an orthotopic system and evaluated the functional relevance of the hyaluronidase Hyal1, a correlate of invasive human prostate cancer, to progression in this model. To provide real-time insights into these processes, we first validated use of an epidermal growth factor-conjugated fluorophore to illuminate orthotopic prostate tumors and their metastases in whole animal imaging. Animals receiving intraprostatic injections were tracked throughout a 6-week period. Tumor sizes were correlated 92% with total fluorescence intensities of 22 prostate tumors. In contrast to the highly tumorigenic and metastatic PC3M-LN4 cells, the 22Rv1 line was orthotopically tumorigenic but not metastatic, despite larger tumor sizes. Lymph node metastasis was successfully imaged in animals with PC3M-LN4 tumors on endpoint dissection. Stable transfection of 22Rv1 cells with Hyal1 did not alter growth kinetics of primary orthotopic tumors, but all animals implanted with Hyal1 transfectants exhibited tumor-positive para-aortic lymph nodes. Hyal1 is implicated as an inducer of prostate cancer metastatic progression.     

Stereocomplex micelle loaded with paclitaxel for enhanced therapy of breast cancer in an orthotopic mouse model

Micelles are promising a nano drug carrier for cancer therapy. However, their application is often limited due to the instability of them in vivo. Herein, we reported the development of stereocomplex micelle (SCM) based on amphiphilic dextran-block-polylactide (Dex-b-PLA) that could improve the stability of micelles, reduce the early release of loaded drugs and target the breast cancer through the enhanced permeability and retention (EPR) effect for enhanced breast cancer therapy. The SCM were fabricated from the equimolar mixture of the enantiomeric Dex-b-PLA copolymers. Paclitaxel (PTX) as a model anti breast cancer drug was loaded in the SCM, noted as SCM/PTX. Transmission electron microscopy (TEM) and dynamic laser scattering (DLS) showed the diameter of SCM/PTX was below100?nm, which was suitable sizes for the EPR effect. The release kinetics of SCM/PTX exhibited that the release of PTX was obviously slow down and showed constant release. In the in vitro antitumor test, the SCM/PTX could effectively suppress the viability of 4T1 cells, which was demonstrated by the MTT assay. Moreover, the SCM/PTX could reduce the distribution of PTX at normal organs and obviously increase the accumulation of PTX at tumor sites. The circulation time of SCM/PTX was also obviously enhanced compared to free PTX. In the in vivo antitumor test, the SCM/PTX effectively inhibited the progression of 4T1 breast cancer in the orthotopic mouse model, as demonstrated by decreased tumor growth and increased apoptosis and necrosis areas within tumor tissues. In addition, the toxic side effects of PTX was also alleviated in the SCM/PTX group. This study introduced a stable micelle system that passive targeted the tumor for enhanced breast cancer therapy.     

In vivo MRI for effective non‐invasive detection and follow‐up of an orthotopic mouse model of lung cancer

One of the main reasons for the dismal prognosis of lung cancer is related to the late diagnosis of this pathology. In this study, we evaluated the potential of optimized lung MRI techniques as a completely non‐invasive approach for non‐small‐cell lung cancer (NSCLC) MRI in vivo detection and follow‐up in a mouse model of lung adenocarcinoma expressing the luciferase gene. Bioluminescent lung tumour cells were orthotopically implanted in immuno‐deficient mice. Ultra‐short echo‐time (UTE) MRI free‐breathing acquisitions were compared with standard gradient‐echo lung MRI (FLASH) using both respiratory‐gated and free‐breathing protocols. The MRI findings were validated against bioluminescence imaging (BLI) and gold‐standard histopathology analysis. Adenocarcinoma‐like pathological tissue was successfully identified in all the mice with gated‐FLASH and non‐gated UTE MRI, and good tumour co‐localization was found between MRI, BLI and histological analyses. An excellent or good correlation was found between the measured bioluminescent signal and the total tumour volumes quantified with UTE MRI or gated‐FLASH MRI, respectively. No significant correlation was found when the tumours were segmented on non‐gated MR FLASH images. MRI was shown to be a powerful imaging tool able to detect, quantify and longitudinally monitor the development of sub‐millimetric NSCLCs. To our knowledge, this is the first study which proves the feasibility of a completely non‐invasive MRI quantitative detection of lung adenocarcinoma in freely breathing mice. The absence of ionizing radiation and the high‐resolution of MRI, along with the complete non‐invasiveness and good reproducibility of the proposed non‐gated protocol, make this imaging tool ideal for direct translational applications. Copyright © 2014 John Wiley & Sons, Ltd.     

Tumor targeting by doxorubicin-RGD-4C peptide conjugate in an orthotopic mouse hepatoma model

Vascular targeting is a novel strategy that directs endothelial toxins at tumor vessels expressing specific markers and kills tumor cells by vascular occlusion. Integrin-binding RGD motif has been reported to have a homing property to experimental tumor vasculature. In the present study, we evaluated the effect of vascular targeting by doxorubicin-RGD-4C conjugate in an orthotopic murine hepatoma model. MTT assay showed that dox-RGD-4C conjugates had lower cytotoxicity against MH134 mouse hepatoma cells than free dox. When given intravenously to mice with implanted orthotopic hepatoma, however, the dox-RGD-4C suppressed the growth of hepatoma more effectively than free dox (mean tumor volumes 24 mm(3) vs. 67 mm(3), respectively; p=0.047). Histologic analysis of the hepatoma tissue revealed prominent tumor cell death in the dox-RGD-4C treated group and complete tumor cell necrosis in 40% of cases. Immunochemical staining showed expression of integrin alphav mainly around the tumor nodule. These results show that dox-RGD-4C conjugate has a better antitumor effect in an orthotopic mouse hepatoma model by tumor targeting. Integrin alphav of hepatoma feeding vessels is suggested to be targeted by the dox-RGD-4C conjugate.     

A peroxide-induced inflammation model for drug testing

Peroxide-induced cellular injury is an important mediator of inflammation. Whether the injection of glucose oxidase (GO) with its generation of H₂O₂ may be utilized as an inflammatory model was studied. Anesthesized mice were injected in the right hind foot with either water or an equal volume of 10 or 100 units/ml GO. The animals were sacrificed at 1.5 or 24 hours post injection and both hind feet amputated and each foot weighed. Difference in weights for each animal's feet was called edema index (EI). GO produced significant increases in weight. The use of GO to produce inflammation may be useful in testing potential peroxide quenching agents.     

An orthotopic mouse model of small cell lung cancer reflects the clinical course in patients

Small cell lung cancer (SCLC) is a highly aggressive subtype of lung cancer with very poor prognosis due to early metastatic spread and development of chemoresistance. In the last 30 years the study of SCLC has been constrained by a lack of primary human tumor specimen thus highlighting the need of a suitable mouse model. In this article we present the establishment of an orthotopic xenograft mouse model which accurately reproduced the clinical course of SCLC. Orthotopic implantation enabled engraftment of primary lung tumors in all injected mice. Furthermore, immunodeficiency of mice allowed formation of spontaneous metastases in characteristic organs. Bioluminescence Imaging, Magnetic Resonance Imaging and Positron emission tomography were applied to monitor engraftment, metabolism and the exact growth of tumors over time. In order to mimic the extensive disease stage, mice were injected with aggressive human chemoresistant cells leading to development of chemoresistant tumors and early metastatic spread. As a proof of concept treatment of tumor-bearing mice with conventional chemotherapeutics reduced tumor volumes, but a complete regression of tumors was not achieved. By mimicking the extensive disease stage our mouse model can facilitate the study of mechanisms contributing to chemoresistance and metastasis formation, as well as drug screening and evaluation of new treatment strategies for SCLC patients.     

An in vitro model for testing biocompatibility of endodontic materials to bone

Calvaria from 6-day old mice labelled 4 days previously with ⁴⁵CaCI₂ were divided into test and control halves and each half cultured separately in vitro. Eluents from four endodontic materials, endomethasone, zinc oxide/eugenol, AH26 and gutta-percha were added separately to the culture medium of each test half. After 24 and 48 h culturing periods, the ⁴⁵CaCI₂ in the media and calvaria was measured by a standard liquid scintillation counting technique and a resorption ratio between test and control halves was computed. This ratio, based on cell mediated resorption, was an indication of toxicity of soluble components of each endodontic material. In accordance with the literature, endomethasone was found, with our method, to be the most toxic and gutta-percha the least toxic of the materials tested. This shows that our model can be used to test the toxicity of other biomaterials to bone cells in vitro.     

A model for testing recombinogenic sequences in the mouse germline

Homologous recombination is a conserved process of genetic exchange generated by homologous pairing of nucleotides. Species diversity and gene evolution are dependent on the outcomes of recombination during germ cell development, yet systems to study mammalian germline recombination, especially those with applications to human genetics, are not well developed. We report on a transgenic mouse system designed to study recombination within test sequences in the male germline utilizing an intron-interrupted lacZ reporter gene. beta-galactosidase positive sperm are detected and quantitated by flow cytometry using fluorogenic substrates. Examination of recombination within a 1.7 kb repeat of test sequences derived from the human glycophorin breakpoint cluster region detects approximately 0.04-0.09% fluorescent sperm. Confirmation that these sperm result from recombination in the germline comes from histochemical staining of testicular cells, examination of spliced mRNA, and PCR analysis of sorted sperm populations. The system is readily adaptable to studies of other sequences reported to have elevated levels of recombination, including those implicated in human genetic disease. Investigations of the molecular basis for genomic instability at specific chromosomal locations may yield important insights into mechanisms of chromosomal loss and rearrangements.      

Intravenous KITENIN shRNA injection suppresses hepatic metastasis and recurrence of colon cancer in an orthotopic mouse model

KITENIN (KAI1 C-terminal interacting tetraspanin) promotes invasion and metastasis in mouse colon cancer models. In the present study, we evaluated the effects of KITENIN knockdown by intravenous administration of short hairpin RNAs (shRNAs) in an orthotopic mouse colon cancer model, simulating a primary or adjuvant treatment setting. We established orthotopic models for colon cancer using BALB/c mice and firefly luciferase-expressing CT-26 (CT26/Fluc) cells. Tumor progression and response to therapy were monitored by bioluminescence imaging (BLI). In the primary therapy model, treatment with KITENIN shRNA substantially delayed tumor growth (P = 0.028) and reduced the incidence of hepatic metastasis (P = 0.046). In the adjuvant therapy model, KITENIN shRNA significantly reduced the extent of tumor recurrence (P = 0.044). Mice treated with KITENIN shRNA showed a better survival tendency than the control mice (P = 0.074). Our results suggest that shRNA targeting KITENIN has the potential to be an effective tool for the treatment of colon cancer in both adjuvant and metastatic setting.     

新型可降解性镁金属合金的骨组织反应评价

目的 探讨组织工程用镁金属合金在大鼠体内的降解过程. 方法 1月龄SD大鼠15只,麻醉后股骨干钻孔,植入表面修饰后的镁合金金属棒,术后皮下注射庆大霉柰预防感染并每周皮下注射钙黄绿素.术后6、9、15、26和80周分别随机处死3只大鼠,并取出植入有金属棒的股骨,各组材料经体视显微镜、光镜、扫描电镜、倒置荧光显微镜观察并行能量色散谱元素分析. 结果 体视显微镜及光镜下观察,可见6、9、15、26周大鼠股骨髓腔中金属移植物周围包裹有新生骨,接触髓腔的金属界面受到不同程度的侵蚀,且侵蚀或降解程度随取材时间的延长而加大.倒置荧光显微镜下观察,可见移植物周围有新骨生成.能量色散谱元素分析表明,含有碳、氮、氧元素的基底膜层在金属移植物周围形成.术后80周大鼠股骨上,植入的金属材料全部降解,基底膜层消失,新生骨层裂解并逐渐被改造吸收. 结论 含有锌和锰元素的镁合金是一种新型体内可降解性金属合金,对体内诱导骨生成有良好作用.     

A novel orthotopic model of breast cancer metastasis to bone

Breast cancer affects approximately one woman in twelve and kills more women than any other cancer. If detected early, patients have a five year survival rate of 66%, but once metastatic disease has developed, there is no effective treatment. About 70% of patients with metastatic disease have bone involvement, while lungs and liver are the other common targets. Bone metastases cause severe pain, pathological fractures and hypercalcaemia and thus are a significant clinical problem. The development of new therapies for metastatic breast carcinoma depends on a better understanding of the mechanism of homing of the tumour cells to bone, liver and lungs and the factors required for their growth in these organs. Research on mechanisms of breast cancer metastasis, particularly to bone, has relied on in vitro studies or on tumour models in which the inoculation route is designed to promote delivery of tumour cells to a specific organ. Metastases in bone are achieved by inoculation into the right ventricle of the heart. To our knowledge there has been no report of a model of metastatic spread from the mammary gland to distant sites which reliably includes bone. In this paper, we describe our recent development of a novel murine model of metastatic breast carcinoma. The new model is unique in that the pattern of metastatic spread closely resembles that observed in human breast cancer. In particular, these murine breast tumours metastasise to bone from the primary breast site and cause hypercalcaemia, characteristics not normally found in murine tumours, but common in human disease. Furthermore, in a preliminary characterisation of this model, we show that secretion of parathyroid hormone-related protein, a role for which has been implicated in breast cancer spread to bone, correlates with metastasis to bone. This model therefore provides an excellent experimental system in which to investigate the factors that control metastatic spread of breast cancer to specific sites, particularly bone. The special advantage of this system is that it involves the whole metastasis process, beginning from the primary site. Existing models consider mechanisms that pertain to growth of tumour once the site has been reached. An understanding of the regulation of these factors by potential therapeutic agents could lead to improvement in therapies designed to combat metastatic disease. For the first time, this development will allow exploration of the molecular basis of site-specific metastasis of breast cancer to bone in a clinically relevant model.     

Anti-TNF effect of combined pravastatin and cilostazol treatment in an in vivo mouse model

Objectives: Pravastatin and cilostazol are used as lipid-lowering and antiplatelet agents, respectively. Regarding their well-known anti-inflammatory effects, the additive effect of the two drugs on anti-TNF functions has not yet been investigated. In the present investigation, the beneficial effect of combined pravastatin and cilostazol on their anti-TNF activities was assessed using an in vivo mouse model.

Methods: Mice were pretreated with pravastatin and/or cilostazol (40?mg/kg of each), orally once two hour prior to an LPS (5?mg/kg, i.p.) challenge. One hour post challenge, blood and descending aorta were collected for serum TNF levels and immune cell infiltration analyses. For survival analysis, pravastatin and/or cilostazol (40?mg/kg of each) were administered 30?minutes prior to d-galactosamine administration (700?mg/kg, i.p.) and TNF (10 µg/kg, i.p.) challenge and mice survival was monitored. We also examined the effect of either drug or the combination of drugs on TNF-mediated MAPK and NF-κB signaling, using Western blot analysis.

Results: Combined treatment of pravastatin and cilostazol significantly decreased serum TNF release and immune cell infiltration in the descending aorta following LPS administration, compared to each single treatment. Additionally, the combined drugs significantly decreased TNF-mediated mouse mortality and downregulated TNF-induced MAPK and NF-κB activation.

Conclusions: These findings suggest that combined pravastatin and cilostazol is more effective for reducing TNF-driven inflammation through their anti-TNF activity than monotherapy.     

An orthotopic skull base model of malignant meningioma

Meningioma tumor growth involves the subarachnoid space that contains the cerebrospinal fluid. Modeling tumor growth in this microenvironment has been associated with widespread leptomeningeal dissemination, which is uncharacteristic of human meningiomas. Consequently, survival times and tumor properties are varied, limiting their utility in testing experimental therapies. We report the development and characterization of a reproducible orthotopic skull-base meningioma model in athymic mice using the IOMM-Lee cell line. Localized tumor growth was obtained by using optimal cell densities and matrigel as the implantation medium. Survival times were within a narrow range of 17-21 days. The xenografts grew locally compressing surrounding brain tissue. These tumors had histopathologic characteristics of anaplastic meningiomas including high cellularity, nuclear pleomorphism, cellular pattern loss, necrosis and conspicuous mitosis. Similar to human meningiomas, considerable invasion of the dura and skull and some invasion of adjacent brain along perivascular tracts were observed. The pattern of hypoxia was also similar to human malignant meningiomas. We use bioluminescent imaging to non-invasively monitor the growth of the xenografts and determine the survival benefit from temozolomide treatment. Thus, we describe a malignant meningioma model system that will be useful for investigating the biology of meningiomas and for preclinical assessment of therapeutic agents.     

In vivo mouse imaging and spectroscopy in drug discovery

Imaging modalities such as micro-computed tomography (micro-CT), micro-positron emission tomography (micro-PET), high-resolution MRI, optical imaging, and high-resolution ultrasound have become invaluable tools in preclinical pharmaceutical research. They can be used to non-invasively investigate, in vivo, rodent biology and metabolism, disease models, and pharmacokinetics and pharmacodynamics of drugs. The advantages and limitations of each approach usually determine its application, and therefore a small-rodent imaging laboratory in a pharmaceutical environment should ideally provide access to several techniques. In this paper we aim to illustrate how these techniques may be used to obtain meaningful information for the phenotyping of transgenic mice and for the analysis of compounds in murine models of disease.     

Evaluation of the genotoxic effects of pyrimethamine, an antimalarial drug, in the in vivo mouse

Pyrimethamine is an antimalarial drug and a known teratogenic agent. With this drug, positive and negative results have been reported by various investigators in in vivo and in vitro genotoxicity/mutagenicity assays. In this investigation the genotoxic effects of pyrimethamine (PY) were tested in mice in vivo systems, using the bone marrow micronucleus test (MNT) and the transplacental MN test (TMNT). PY at the highest dose (40 mg/kg) induced statistically significant MN in bone marrow cells at 24 and 48 h. In the transplacental MN test, PY did not induce significant MN in fetal liver or in maternal bone marrow. Teratogenesis Carcinog. Mutagen. 20:65-71, 2000.     

Microstructure of trabecular bone in a mouse model for down syndrome

Down syndrome (DS) is caused by trisomy of human chromosome 21 (Hsa21) and results in a suite of dysmorphic phenotypes, including effects on the postcranial skeleton and the skull. We have previously demonstrated parallels in the patterns of craniofacial dysmorphology in DS and in the Ts65Dn mouse model for DS. The specific mechanisms underlying the production of these changes in craniofacial shape remain unknown. High‐resolution computed tomography scan data were collected for the presphenoid bone of euploid and aneuploid mice. Three‐dimensional morphometric parameters of trabecular bone were quantified and compared between euploid and aneuploid mice using nonparametric statistical tests. Aneuploid presphenoid bones were smaller than those of their euploid littermates and had lower bone volume fraction and fewer, more rod‐like trabeculae. The differences in cancellous bone structure suggest that bone development, perhaps including bone modeling and remodeling, is affected by aneuploidy. These differences may contribute to the observed dysmorphology of skull and postcranial skeletal phenotypes in DS. Anat Rec, 2007. © 2007 Wiley‐Liss, Inc.     

Real-time in vivo green fluorescent protein imaging of a murine leishmaniasis model as a new tool for Leishmania vaccine and drug discovery

Leishmania species are obligate intracellular protozoan parasites that cause a broad spectrum of clinical diseases in mammalian hosts. The most frequently used approach to quantify parasites in murine model systems is based on thickness measurements of the footpad or ear after experimental infection. To overcome the limitations of this method, we used a Leishmania mutant episomally transfected with enhanced green fluorescent protein, enabling in vivo real-time whole-body fluorescence imaging, to follow the progression of Leishmania infection in parasitized tissues. Fluorescence correlated with the number of Leishmania parasites in the tissue and demonstrated the real-time efficacy of a therapeutic vaccine. This approach provides several substantial advantages over currently available animal model systems for the in vivo study of immunopathogenesis, prevention, and therapy of leishmaniasis. These include improvements in sensitivity and the ability to acquire real-time data on progression and spread of the infection.     

Immunohistochemical localization of steroid receptor coactivators in chondrosarcoma: An in vivo tissue microarray study

Chondrosarcoma is the second most common type of primary bone malignancy following up osteosarcoma, characterized by resistance to conventional chemotherapeutic agents and radiation regimens. The p160 family members steroid receptor coactivator-1 and -3 (SRC-1 and SRC-3) have been implied in the regulation of cancer growth, migration, invasion, metastasis and chemotherapeutic resistance; but we still lack detailed information about the levels of SRCs in chondrosarcoma. In this study, expression of SRC-1 and SRC-3 in chondrosarcoma was examined by immunohistochemistry with tissue microarrays; the four score system (0, 1, 2 and 3) was used to evaluate the staining. The results showed that there were no gender-, site- or age-differences regarding the expression of SRC-1 or SRC-3 (p > 0.05); organ (bone or cartilage) -differences were only detected for SRC-1 but not SRC-3 (p < 0.05). Significant higher levels of SRC-1 and SRC-3 were detected in MDC and PDC when compared to WDC. Our study clearly demonstrated differentiation-dependant expression of SRC-1 and SRC-3 in chondrosarcoma, may be novel targets for the prognosis and/or treatment of chondrosarcoma, would have opened a new avenue and established foundation for studying chondrosarcoma.     

Nitrogen mustard drug resistant B-cell chronic lymphocytic leukemia as an in vivo model for crosslinking agent resistance

Acquired resistance is a limiting factor in chemotherapy. We have employed nitrogen mustard resistant B-cell chronic lymphocytic leukemia (B-CLL) as a clinically relevant model to study this phenomenon. Resistance in B-CLL is associated with enhanced repair of nitrogen mustard crosslinks. In order to identify the repair pathway responsible for nitrogen mustard resistance, lymphocytes were screened for cross-resistance to a variety of DNA damaging agents. The MTT assay was used to measure the resistance of B-CLL lymphocytes to various DNA damaging agents, including nitrogen mustards, UV light, methyl methanesulfonate, and mitomycin C. We have shown that B lymphocytes from patients with nitrogen mustard resistant chronic lymphocytic leukemia reflect their clinical status. This assay allows us to classify lymphocytes as nitrogen mustard sensitive or resistant, based on in vitro observations. The resistant population was 5.6 and 4.1 fold more resistant to the nitrogen mustard analogs, chlorambucil and melphalan, respectively. Resistant lymphocytes displayed no increased resistance to either methyl methanesulfonate or UV light, indicating that neither classical base nor nucleotide excision repair is rate-limiting in resistance. Resistant lymphocytes were 6.0 and 2.2 fold more resistant to mitomycin C and cis-diamminedichloroplatinum (II), respectively, suggesting enhanced crosslink repair. Neither glutathione nor glutathione S-transferase levels correlated with resistance. The development of nitrogen mustard drug resistance in B-CLL appears to be associated with cross-resistance to other bifunctional alkylating agents which produce interstrand crosslinks. Our results indicate that resistance to nitrogen mustards in chronic lymphocytic leukemia is associated with enhanced repair of DNA crosslinks which may involve a recombination dependent system. This model should prove very useful in the elucidation of the molecular mechanisms of crosslink repair.