DNA damage emergency: cellular garbage disposal to the rescue?

The proteasome is a cellular machine found in the cytosol, nucleus and on chromatin that performs much of the proteolysis in eukaryotic cells. Recent reports show it is enriched at sites of double-stranded DNA breaks (DSBs) in mammalian cells. What is it doing there? This review will address three possibilities suggested by recent reports: in degrading proteins after their ubiquitination at and eviction from chromatin; as a deubiquitinase, specific to the antagonism of ubiquitin conjugates generated as part of the signalling of a DSB; and as a functional component of DNA repair mechanism itself. These findings add complexity to the proteasome as a potential therapeutic target in cancer treatment.

The Forkhead Box M1 protein regulates BRIP1 expression and DNA damage repair in epirubicin treatment.

FOXM1 is implicated in genotoxic drug resistance but its role and mechanism of action remain unclear. Here, we establish that γH2AX foci, indicative of DNA double-strand breaks (DSBs), accumulate in a time-dependent manner in the drug-sensitive MCF-7 cells but not in the resistant counterparts in response to epirubicin. We find that FOXM1 expression is associated with epirubicin sensitivity and DSB repair. Ectopic expression of FOXM1 can increase cell viability and abrogate DSBs sustained by MCF-7 cells following epirubicin, owing to an enhancement in repair efficiency. Conversely, alkaline comet and γH2AX foci formation assays show that Foxm1-null cells are hypersensitive to DNA damage, epirubicin and γ-irradiation. Furthermore, we find that FOXM1 is required for DNA repair by homologous recombination (HR) but not non-homologous end joining (NHEJ), using HeLa cell lines harbouring an integrated direct repeat green fluorescent protein reporter for DSB repair. We also identify BRIP1 as a direct transcription target of FOXM1 by promoter analysis and chromatin-immunoprecipitation assay. In agreement, depletion of FOXM1 expression by small interfering RNA downregulates BRIP1 expression at the protein and mRNA levels in MCF-7 and the epirubicin-resistant MCF-7 Epi(R) cells. Remarkably, the requirement for FOXM1 for DSB repair can be circumvented by reintroduction of BRIP1, suggesting that BRIP1 is an important target of FOXM1 in DSB repair. Indeed, like FOXM1, BRIP1 is needed for HR. These data suggest that FOXM1 regulates BRIP1 expression to modulate epirubicin-induced DNA damage repair and drug resistance.

Biallelic mutation of MSH2 in primary human cells is associated with sensitivity to irradiation and altered RAD51 foci kinetics.

BACKGROUND: Reports of differential mutagen sensitivity conferred by a defect in the mismatch repair (MMR) pathway are inconsistent in their conclusions. Previous studies have investigated cells established from immortalised human colorectal tumour lines or cells from animal models. METHODS: We examined primary human MSH2-deficient neonatal cells, bearing a biallelic truncating mutation in MSH2, for viability and chromosomal damage after exposure to DNA-damaging agents. RESULTS: MSH2-deficient cells exhibit no response to interstrand DNA cross-linking agents but do show reduced viability in response to irradiation. They also show increased chromosome damage and exhibit altered RAD51 foci kinetics after irradiation exposure, indicating defective homologous recombinational repair. DISCUSSION: The cellular features and sensitivity of MSH2-deficient primary human cells are broadly in agreement with observations of primary murine cells lacking the same gene. The data therefore support the view that the murine model recapitulates early features of MMR deficiency in humans, and implies that the variable data reported for MMR-deficient immortalised human cells may be due to further genetic or epigenetic lesions. We suggest caution in the use of radiotherapy for treatment of malignancies in individuals with functional loss of MSH2.

Influence of buried hydrogen-bonding groups within monolayer films on gas-surface energy exchange and accommodation.

Self-assembled monolayers (SAMs) of carbonyl-containing alkanethiols on gold are employed to explore the influence of hydrogen-bonding interactions on gas-surface energy exchange and accommodation. H-bonding, COOH-terminated SAMs are found to produce more impulsive scattering and less thermal accommodation than non-H-bonding, COOCH3-terminated monolayers. For carbamate-functionalized SAMs of the form Au/S(CH2)16OCONH(CH2)(n-1)CH3, impulsive scattering decreases and accommodation increases as the H-bonding group is positioned farther below the terminal CH3.

Mouse ST6Gal sialyltransferase gene expression during mammary gland lactation.

The sialyltransferase ST6Gal mediates the biosynthetic addition of sialic acid, via an alpha2,6 linkage, to the nonreducing end of terminal lactosamine structures. Transcription of the murine ST6Gal gene, Siat1, is regulated by the selective use of multiple promoters in a tissue- and development-specific manner. Here we report that Siat1 mRNA expression is dramatically elevated in lactating (relative to virgin) mouse mammary gland. The predominant ST6Gal mRNA species expressed in lactating mammary gland is a heretofore undocumented isoform containing a unique 5'-untranslated region originating from the mouse Siat1 genetic region, now defined as Exon L, residing 549-bp 5' of the previously characterized Exon X(2). Thus, the novel ST6Gal mRNA form initiates transcription from the region designated as p4 and incorporates the unique sequence from Exon L in 5'-juxtaposition to commonly shared sequences encoded on Exon I to Exon VI. In contrast, cells derived from virgin mammary tissue expressed only the housekeeping mRNA form derived from p3, with Exon O sequence preceding Exons I-VI. The Exon L-containing, p4 class of mRNA was also not detected in a survey of eight other mouse tissues. Previous reports have indicated a strong correlation between mammary cancers and elevated ST6Gal expression in rats and in human patients. However, we uncovered neither elevated expression of ST6Gal mRNA nor appearance of p4 class in mouse breast carcinomas experimentally induced by transformation with the polyoma-middle T oncogene. A number of established breast carcinoma cell lines were also examined, with ST6Gal mRNA and activity generally low. Moreover, with the exception of the Shionogi cell line, p4 class of ST6Gal mRNA was not expressed in any of the mouse breast carcinoma specimens examined. Taken together, our data indicate that murine ST6Gal induction during lactation is achieved by de novo recruitment of a normally silent promoter. Furthermore, the data provide no support for elevated Siat1 expression on the mRNA level in association with murine mammary gland carcinogenesis. With the single exception of the Shionogi cell line, the p3 class remains the predominant ST6Gal mRNA expressed in all other murine mammary carcinoma cells examined.

The Sp1 transcription factor regulates cell type-specific transcription of MUC1.

Because the MUC1 mucin is highly expressed in breast and other carcinomas, interest is focused on the MUC1 promoter, particularly in the context of the delivery of genes to carcinomas. Earlier in vitro studies showed that the region between -152 and -66 of the MUC1 promoter is required for transcriptional activity in MUC1-expressing cells. Experiments reported here showed that sequences -119/-62 within this region are able to modulate transcription of the heterologous constitutively active herpes simplex virus thymidine kinase promoter in a pattern consistent with MUC1 expression. Band-shift experiments showed that although several factors (including Sp1 and Sp3) bind to these sequences, the element important in directing this MUC1 pattern of expression was an Sp1 GC box at -97. The data also show that the positioning or phase of the GC box was crucial for directing expression. The importance of the Sp1 transcription factor was confirmed by demonstrating that overexpression of Sp1 in MUC1-nonexpressing cells increased, not only the expression of a reporter gene driven by the 1.4-kb MUC1 promoter, but also the expression of MUC1 from the endogenous gene. Together, these data define an important role for Sp1 in the cell type-specific transcription of MUC1.

Up-regulation of MUC1 in mammary tumors generated in a double-transgenic mouse expressing human MUC1 cDNA, under the control of 1.4-kb 5' MUC1 promoter sequence and the middle T oncogene, expressed from the MMTV promoter.

In this study we examined the regulation of expression of the human MUC1 gene in vivo, by developing MUC1 transgenic mice. The data showed that epithelial-specific expression of MUC1 can be directed by just 1.4 kb of 5' flanking sequence using MUC1 cDNA as a reporter gene in vivo. Furthermore, high levels of MUC1 expression were seen in the lactating mammary gland and in spontaneous mammary tumors generated by crossing the MUC1 transgenics with mice transgenic for the polyoma middle T oncogene under the control of the mouse mammary tumor virus promoter. This pattern of expression in epithelial tissues is comparable to the expression of MUC1 in humans and also to the expression pattern in another transgenic mouse line developed with a 10.6-kb genomic MUC1 fragment. This study confirmed that MUC1 is a compact gene and demonstrated that the 1.4-kb 5' sequence not only directs epithelial-specific expression of MUC1 in vivo but also contains the elements governing the up-regulation observed during lactation and in malignancy.

Effects of blast exposure on exercise performance in sheep.

BACKGROUND: The effects of blast on maximal exercise performance were investigated in sheep that were trained to perform maximal exercise. METHODS AND RESULTS: Sheep were fully instrumented for determination of pulmonary and systemic hemodynamics. Blast exposure was administered by using a compressed air driven shock tube that was positioned to primarily produce cardiopulmonary injury. Four levels of exposure were used that were known to produce sublethal injury ranging from little or no grossly observable cardiopulmonary injury (level 1) to confluent ecchymosis of the heart, lung, or both (level 4). We evaluated maximal exercise performance 1 hour after exposure to level 1, level 2, and level 3 and 24 hours after level 3 and level 4. VO2max was not significantly decreased 1 hour after exposure to level 1 but was decreased after exposure to level 2 (29.9%) and level 3 (49.3%). Significant improvement in exercise performance was observed in 24 hours, as VO2max was not significantly decreased 24 hour after level 3. VO2max was decreased 24 hour after level 4 injury (30.8%). CONCLUSION: Cardiovascular data collected during exercise suggested that acute cardiopulmonary injury is responsible for the exercise performance decrement observed 1 hour after exposure and that significant recovery of function is observed 24 hours after blast injury.

Expression patterns of the human papillomavirus type 16 transcription factor E2 in low- and high-grade cervical intraepithelial neoplasia.

Specific antibodies against the C-terminus of E2, produced by affinity purification of polyclonal antisera, have been used to identify the cellular populations which express the HPV 16 E2 transcription factor, in a series of formalin-fixed, paraffin-embedded cervical tissues. Cases were selected for both the presence of HPV 16 DNA (confirmed by multiple gene-specific PCR detections) and the presence of multiple grades of cervical intraepithelial neoplasia (CIN). The data indicate that E2 expression is highest in CIN I and in koilocytic lesions. Lower expression was observed in CIN II and little in CIN III lesions. In contrast, there was some restoration of E2 expression in invasive carcinomas, although the intracellular distribution was much more diffuse. The location of E2 expression to the superficial layers of the cervical epithelium, as well as the occurrence of some basal expression in CIN I, suggests that antibodies against HPV 16 E2 could be a useful adjunct to standard histological techniques for the detection of 'at-risk' patients as part of a cervical screening programme.

Cardiopulmonary effects of high-impulse noise exposure.

In high-energy impulse noise environments, the biomechanical coupling process between the external forces and the pathophysiology of cardiopulmonary injury is not well understood. A 12-in-diameter compressed air-driven shock tube with reflector plate was used to induce three levels of pulmonary contusion injury in a large animal model. Twenty-one anesthetized sheep were exposed to the various levels of impulse noise generated by the shock tube, with six additional sheep serving as a control group. Pathologic evaluations, performed 3 hours after exposure, showed pulmonary contusion ranging from minor petechial changes on the surface of the lung parenchyma to diffuse ecchymoses affecting as much as 60% of the lung. The gross pathologic observations of injury produced by exposure to the impulse noise produced by the shock tube were similar to those reported for blunt impact trauma or exposure to chemical or grain-dust explosions. The extent of lung injury (lung injury index) was quantitatively assessed. A semilogarithmic relationship between the lung injury index and the measured peak pressure was demonstrated. A significant linear correlation was demonstrated between lung injury index and lung weight-to-body weight ratio. Significant cardiopulmonary changes were also observed as a result of exposure to high-impulse noise. Although in most cases the degree of change was related to the severity of the injury, significant cardiopulmonary function changes were also observed in the absence of significant grossly observable pulmonary injury. Cardiac injury was indicated by decreased cardiac output and hypotension at all levels of injury and might be the result of myocardial contusion or air emboli. Pulmonary injury was demonstrated by respiratory acidosis, increases in lung resistance, and decreases in lung compliance and lung volume. Arterial PO2 appeared to be the most sensitive parameter of injury and was decreased for all measurement intervals for all exposure groups.

The use of the Ilizarov technique in the correction of lower extremity deformities in children.

The first time the Ilizarov technique was used at Children's Hospital of Los Angeles, 25 limb segments were treated in 15 patients. Thirteen segments (52%) required angular correction, 6 (24%) required lengthening, and 6 (24%) required both angular correction and lengthening. Average angular correction was 21 degrees (8 degrees to 41 degrees); average length gained was 44 mm (25 to 85 mm). All patients except two achieved preoperative goals. A total of 17 minor (.68/limb) and four major (.16/limb) complications occurred in the 25 limb segments in the study. The total complication rate was .84 complications per limb segment. The most common complication was pin tract infection (11 patients). This comprised 52% of complications and occurred in 44% of limb segments.

Anterior-posterior stability in partially conforming condylar knee replacement.

The purpose of the study was to estimate the anterior-posterior displacements that would occur with total knee prosthetic designs of different sagittal radii under functional conditions. A key question was whether some designs provided excessive constraint. Patients who had different total knee prosthetic designs were tested in a special rig where the AP drawer was measured under loaded and unloaded conditions. These same designs of total knee prostheses were then tested in a rig under different conditions of compressive and shear loads. Using these experiments as verification, a theoretical treatment was developed to predict the AP displacement for different activities. It was concluded that designs with low to moderate conformity required soft tissue restraint under more strenuous activities, or where high shear forces occurred under low compressive loads. Designs of moderate to high conformity could be fully self-stabilizing under all realistic conditions. However, for all designs, the AP displacement was only a few millimeters under functional loads, indicating that special care must be taken during surgery to avoid conflict between the condylar surfaces and the retained cruciate ligaments so that restriction of motion and excessive wear do not occur.

Nonauditory injury threshold for repeated intense freefield impulse noise.

Exposure to impulse noise is an important occupational health concern. The risk of injury to auditory structures is well recognized and provides the cornerstone for present safety standards. For freefield impulse noise, nonauditory injury is dependent on peak pressure, positive phase duration (or impulse), and number of exposures. Trivial laryngeal petechiae are shown to precede nonauditory injury to more critical organs (ie, pulmonary and gastrointestinal systems). This study identifies the critical impulse noise thresholds causing trivial laryngeal petechial changes resulting from exposure to 5, 25, and 100 repetitions of specific levels of impulse noise. Because of anatomical differences, sheep should be slightly more susceptible to impulse noise laryngeal petechial changes than man; therefore, it seems reasonable to set the absolute limits for human occupational exposure levels below those causing laryngeal petechiae in sheep for persons wearing adequate hearing protection. This study does not address human auditory injury that may occur above or below these exposure limits even with proper hearing protection.

Tissue welding using a low energy microsurgical CO2 laser.

A low energy, CO2 laser adapted specifically for microsurgery can be used to assist in anastomosis by "welding" tissue through thermal coagulation. Which tissue constituents contribute to the bond are not known, and different constituents may be important in welding different organs.

Primary malignant fibrous histiocytoma of the lung. Fine needle aspiration cytologic features.

A case of primary malignant fibrous histiocytoma of the lung occurring in a 71-year-old woman is presented. The preoperative aspiration cytology showed a large-cell, undifferentiated, malignant neoplasm suggestive of carcinoma. Subsequent histologic examination revealed a primary malignant fibrous histiocytoma. The diagnosis was confirmed by electron microscopic and immunohistochemical studies. Cytologic features of this rare primary pulmonary sarcoma are discussed.

Carbon dioxide laser use in wound sealing and epikeratophakia.

This paper presents current clinical and experimental uses of the carbon dioxide laser and the results of our human and animal tissue fusion investigations. Trials were conducted with human scleral and corneal eye bank tissue and in albino rabbits. With power settings of 100 to 200 mW, optimal laser effect consisting of slight whitening and minimal edge shrinkage occurred between 10 and 70 mJ of total energy. No tissue adherence could be demonstrated with the parameters tested. A final study was made on the feasibility of sealing an epikeratophakia lenticle to its recipient bed using the carbon dioxide laser. The average force required to remove the button in the control experiments was 6.45 g and this increased to 6.99 g after laser treatment although no fusion was apparent. This was statistically significant (P less than .05).

Laser-assisted microsurgical anastomosis.

A low power carbon dioxide laser was used to perform 212 end-to-end laser-assisted microvascular anastomoses (LAMA) of femoral arteries (mean diameter, 1.2 mm) in Sprague-Dawley rats. Eighty-two conventional microvascular suture anastomoses (CMSA) utilizing 10-0 monofilament interrupted sutures were done for comparison of techniques and wound healing. The mean duration of each anastomosis procedure was 16 minutes for the LAMA repairs, compared to an average of 27 minutes for the CMSA repairs (P less than 0.05). All anastomoses were patent at the completion of the procedure. Each laser-assisted anastomosis required an average of seven intermittent laser exposures of 0.1 to 0.3 seconds each with approximately 80 mW of CO2 (wavelength = 10.6 micron) radiation at a spot size of 150 micron. A patency rate of 95% was obtained on the LAMA vessels (202 of 212) compared to 96% for the CMSA repairs (79 of 82). A total of 14 aneurysms were noted in the LAMA group (7%) compared to 11 in the CMSA (13%). All aneurysms were in patent vessels. Histological analysis indicates that the progression of wound healing of LAMA and CMSA anastomoses follows similar paths chronologically and morphologically with increased scar tissue formation around the suture. Scanning electron microscopy confirms the comparable luminal healing of the LAMA and CMSA vessels, with complete reendothelialization occurring by 3 weeks postoperatively. The tensile strength of the LAMA repair, although low immediately after operation, is comparable to that of the intact artery at 21 days. These findings suggest that a low energy carbon dioxide microsurgical laser has potential beneficial clinical application for anastomosis of small vessels.

Carbon dioxide laser stapedotomy. Thermal effects in the vestibule.

Small-fenestra stapedotomy has recently been popularized in efforts to reduce the incidence of sensorineural hearing loss following stapes surgery for otospongiosis. Lasers have been advocated as a tool to fenestrate the stapes footplate. Conversion of radiant energy from the laser into heat in the vestibule represents the greatest potential risk to the inner ear. Using a carbon dioxide laser with a focal point of 150 microns at 300 mm, fenestrations of the stapes footplate were performed in a series of 14 anesthetized cats. The laser power output ranged from 0.47 to 3.05 W, with pulse durations of 0.2 and 0.5 s. Resultant temperature elevations in the vestibule, measured by a thin-wire thermocouple, ranged from 0 to 4.4 degrees C (0 to 8 degrees F) and directly correlated to wattage and duration of the laser-beam pulse. Temperature changes could be reduced by use of lower wattage, shorter pulse duration, timed intervals between pulses, and convection cooling of the promontory.