Vijaya Vegesna

Mutations of the CHK2 gene are found in some osteosarcomas, but are rare in breast, lung, and ovarian tumors.

Checkpoint genes, activated in response to DNA damage and other stresses, are frequently targeted for alteration in cancer. Checkpoint kinase 2 (CHK2, CDS1, RAD53) is activated by ataxia telangiectasia mutated (ATM) in response to γ irradiation. Activated CHK2 stabilizes TP53, and acts on other cell cycle and stress regulators. These findings place CHK2 in the middle of a pathway frequently targeted in cancer. Because of this, and the observation that CHK2 mutations are inherited in some Li‐Fraumeni cancer syndrome families, we decided to examine the role of CHK2 mutations in sporadic cancers. Exploiting the genomic sequence of chromosome 22, we looked for mutations in the exons and intron junctions of the CHK2 gene in DNA samples from 170 patients (57 osteosarcomas, 25 other sarcomas, 35 nonsmall‐cell lung, 20 ovarian, and 33 breast cancers). Missense mutations affecting the forkhead and kinase domains were detected in four osteosarcomas and in one ovarian and one lung cancer. These findings of CHK2 gene mutations are consistent with osteosarcoma being a defining tumor of Li‐Fraumeni syndrome. The occurrence of CHK2 mutations in sporadic cancers emphasizes the importance of the stress pathway which includes TP53.

C/EBP-β, C/EBP-δ, PU.1, AML1 genes: mutational analysis in 381 samples of hematopoietic and solid malignancies

Abstract Mutations of transcription factors are associated with the pathogenesis of cancer. Genomic DNA from 381 cancers and cell lines representing leukemias, lymphomas and a variety of solid tumors were examined for mutations of genes coding for the C/EBP-β, C/EBP-α, PU.1, and AML1 transcription factors using single strand conformation polymorphism (SSCP) and direct DNA sequencing. Mutation of C/EBP-β (a chronic myelogenous leukemia cell line, Kcl22) and C/EBP-δ (a Burkitt’s lymphoma cell line, Raji) were found. Interestingly, the sample with a C/EBP-β alterations had two missense (P236L and G252A) and two silent mutations in a highly conserved region of the gene. The C/EBP-δ alteration in Raji was a missense mutation (A177G). These findings suggest that mutations of the C/EBP-β, C/EBP-δ, PU.1, and AML1 rarely contribute to the development of hematopoietic or solid cancers.

The Effect of Local and Systemic Irradiation on Impairment of Wound Healing in Mice

These experiments were designed to quantify the impact of local or systemic irradiation on the healing of full-thickness skin wounds in mice. Mice received total-body hemibody, or skin irradiations, prior to wounding. Wound tensile strength measured on day 14 was used as an end point. The dose to achieve an isoeffect of 40% of the tensile strength of the wounded controls was 13 Gy less for the total-body exposure than for local skin irradiation. Histological observation showed markedly fewer inflammatory cells in the wounded skin sections from total-body and hemibody-irradiated mice compared with those receiving only skin irradiation. These data demonstrate that the healing response in irradiated skin is dependent upon the extent of hematopoietic suppression and that at low doses this determines the outcome rather than damage to fixed target cells such as dermal fibroblasts, which are relatively resistant to radiation.

Aberrant expression of neutrophil and macrophage-related genes in a murine model for human neutrophil-specific granule deficiency

Neutrophil‐specific granule deficiency involves inheritance of germline mutations in the CCAAT/enhancer‐binding protein ε (C/EBPE) gene. Humans and mice lacking active C/EBPε suffer frequent bacterial infections as a result of functionally defective neutrophils and macrophages. We hypothesized that these defects reflected dysregulation of important immune response genes. To test this, gene expression differences of peritoneally derived neutrophils and macrophages from C/EBPε−/− and wild‐type mice were determined with DNA microarrays. Of 283 genes, 146 known genes and 21 expressed sequence tags (ESTs) were down‐regulated, and 85 known genes and 31 ESTs were up‐regulated in the C/EBP−/− mice. These included genes involved in cell adhesion/chemotaxis, cytoskeletal organization, signal transduction, and immune/inflammatory responses. The cytokines CC chemokine ligand 4, CXC chemokine ligand 2, and interleukin (IL)‐6, as well as cytokine receptors IL‐8RB and granulocyte‐colony stimulating factor, were down‐regulated. Chromatin immunoprecipitation analysis identified binding of C/EBPε to their promoter regions. Increased expression for lipid metabolism genes apolipoprotein E (APOE), scavenger receptor class B‐1, sorting protein‐related receptor containing low‐density lipoprotein receptor class A repeat 1, and APOC2 in the C/EBPε−/− mice correlated with reduced total cholesterol levels in these mice before and after maintenance on a high‐fat diet. Also, C/EBPε‐deficient macrophages showed a reduced capacity to accumulate lipids. In summary, dysregulation of numerous, novel C/EBPε target genes impairs innate immune response and possibly other important biological processes mediated by neutrophils and macrophages.

Fitting the linear-quadratic model using time of occurrence as the end-point for quantal response multifraction experiments

A statistical technique is given for fitting the linear-quadratic model to experimental quantal response multifraction data using the time of the response as the end-point. The analysis used is based on the Cox Proportional Hazards model. The technique is useful for late effects where the time of occurrence of the response is dose dependent. The technique is compared to logistic regression analysis and the advantages and disadvantages are discussed. Both methods are applied to a lung pneumonitis experiment and a kidney experiment.

Epilation in mice after single and multifractionated irradiation

The response of the resting (fully formed) hair follicle to irradiation was studied using an arbitrary 6 unit scale of epilation as an endpoint. Dose-response curves for single and multifractionated irradiations were analyzed in terms of the dose that gave a certain response in 50% of the mice (HRD50). HRD50 values increased with decrease in dose per fraction even when changing from 1.6 Gy to 1.15 Gy per fraction. The plots of the reciprocal of isoeffective doses versus dose per fraction are nonlinear suggesting either inappropriateness of the linear-quadratic model over the whole range of doses, or incompleteness of repair given as they were, at 3-h intervals. Allowing for incomplete repair, estimates for the alpha/beta ratio were 3.1 Gy and 1.7 Gy for the complete data set or for doses less than 7 Gy, respectively. The steep slope of the isoeffect curve plotting total dose versus dose per fraction was comparable to late responding normal tissues like lung, kidney and spinal cord. Such a response is consistent with slow proliferation of the matrix cells of resting follicles. The same animals were kept to assess the effect of dose fractionation on lethal injury to the lung. Since epilation occurs well before death from lung injury, the data for the two responses were correlated to determine whether epilation might help in predicting the probability of the later development of lung injury: no association was found.

Role of the Thymus in Radiation-Induced Lung Damage after Bone Marrow Transplantation

Pneumonitis developing after total-body irradiation and bone marrow transplantation can be a serious complication of this form of therapy. In this study, the incidence of lung damage in a murine syngeneic transplant model was found to be decreased by prior removal of the thymus, indicating that thymus-derived cells, even in the absence of complicating factors such as graft-versus-host disease and opportunistic pathogens, can contribute to radiation-induced lung damage. It is suggested that the increased damage is due to a syngeneic graft-versus-host reaction mediated by a regenerating lymphoid system. If this concept is correct, new strategies can be identified that might be employed in altering the incidence of this serious transplant-related complication.

Postoperative Irradiation Impairs or Enhances Wound Strength Depending on Time of Administration

Irradiation can complicate surgical wound healing, yet little is known of the importance of the time between surgery and irradiation on this process. This study investigated the impact of postoperative irradiation on gain in would tensile strength in a murine skin model. Irradiation on the same day as wounding or to 2-day-old wounds reduced wound tensile strength. In contrast, postoperative irradiation delivered at 7, 9 and 14 days transiently enhanced wound tensile strength, as measured 3 but not 4 or 5 weeks later. This effect was independent of the inclusion (hemibody) or exclusion (skin alone) of the hematopoietic system in the field of irradiation. Radiation-enhanced wound tensile strength was greater and occurred earlier after higher radiation doses. Even though the effect of irradiation in enhancing wound tensile strength is transitory, it could be important in assisting early wound healing.

The Effect on Depigmentation after Multifractionated Irradiation of Mouse Resting Hair Follicles

The function of melanocytes, i.e., pigmentation, was studied after doses of radiation given in one to eight fractions ranging from 0.9 to 4.0 Gy by quantifying depigmentation of particular (zig-zag) hairs in resting phase in the mouse. Considerable variability in response was noted, perhaps related to variations in growth status of the hair follicle. The slope of the single-dose survival curve is described by a D0 value of 1.47 Gy over a dose range 5 to 10 Gy. A weighted, nonlinear regression analysis of the multifraction data gave estimates of alpha/beta of 6.5 Gy for the linear quadratic model. The same analysis suggests that there are about four clonogenic melanocytes per hair follicle. There was a fluctuating pattern of recovery in the early hours after exposure to a dose of 4.0 Gy but no evidence of melanocyte regeneration up to 4 days. However, a characteristic of the data was its variability, suggesting that the radiation response of melanocytes over the dose range 0.9 to 10 Gy may be very variable, reflecting, perhaps, variability in the kinetic status of the melanocyte.

Efficacy of I.V. or I.P. injected cytotoxic drugs on proliferating and non-proliferating hair follicles of the mouse☆

A range of drug doses of Adriamycin (ADR), Actinomycin-D (ACT-D), and Mitomycin-C (MMC) were given i.v. or i.p. to mice 1 day after a priming dose of radiation to elicit epilation response. The hair follicles were stimulated through plucking 11 days before irradiation or were unstimulated to represent proliferating and nonproliferating populations. The maximum epilation that appears at 8 days or at 8.5 weeks for proliferating and nonproliferating follicles was quantified using a subjective scale. In general, the i.v. route of administration was more effective than i.p. for all three drugs. Proliferating follicles were more susceptible than non-proliferating follicles to the action of drugs, especially ADR (p = .0001). Radiation doses which would give the same effect as ADR were calculated for proliferating follicles: 8 mg/kg given i.v. was equivalent to 4.6 (3.9, 5.2) Gy. For i.p. administration, 8 mg/kg was equivalent to only 0.6 (-0.1, 1.3) Gy. The in vivo assay of drug effect on hair follicles has advantages over LD10 as a model for toxicological investigation of new drugs: it can assess response of proliferating or non-proliferating cells of the same histotype and, in the case of proliferating follicles, it is quicker, thus enabling the use of doses higher than LD50 for bone marrow deaths.