The hydroxyurea-induced loss of double-minute chromosomes containing amplified epidermal growth factor receptor genes reduces the tumorigenicity and growth of human glioblastoma multiforme.

OBJECTIVE: We investigated whether the hydroxyurea-induced loss of double-minute chromosomes containing amplified epidermal growth factor receptor (EGFR) genes would lead to a loss of tumorigenicity of a glioblastoma multiforme cell line. METHODS: Glioblastoma multiforme cells were treated in vitro with 0 (HU0) or 100 micromol/L (HU100) hydroxyurea and then injected into the flanks of nude mice. Survival and tumor volumes were evaluated. Pulsed-field gel electrophoresis, Southern blot hybridization, and slot-blot analysis were used to determine EGFR amplification levels. Flow cytometry and immunofluorescent staining were used for cell-cycle analysis and EGFR protein expression. RESULTS: Prior to injection, HU100 cells lost 95% of their amplified EGFR genes and developed into tumors 6 weeks after injection versus 3 weeks for HU0 cells. Mice with HU100 tumors had a median survival of 62 days versus 43 days for control mice with HU0 tumors. Pulse-field gel electrophoresis analysis showed that HU100 tumors had reamplified the EGFR gene as double-minute chromosomes of the same size as those originally present before hydroxyurea treatment. When HU100 cells were cultured in the absence of hydroxyurea, the EGFR gene also reamplified. HU100 cells grew at less than half the rate of untreated HU0 control cells in culture and showed a decreased number of cells entering the cell cycle. Immunofluorescent staining of HU150 (150 micromol/L) cells showed decreased EGFR protein expression. CONCLUSION: The EGFR gene is important for tumorigenicity in mice and growth in culture. Hydroxyurea induces the loss of double-minute chromosome-amplified EGFR genes against a selection gradient and significantly delays the onset of tumors. These results support the potential use of low-dose hydroxyurea for the treatment of human glioblastoma multiforme.

The scid defect results in much slower repair of DNA double-strand breaks but not high levels of residual breaks.

Severe combined immune deficiency (scid) mice fail to produce mature B and T cells and are sensitive to ionizing radiation. They contain a mutation in the 460-kDa catalytic subunit of the DNA-dependent protein kinase that is involved in both V(D)J rejoining and DNA double-strand break (DSB) repair. The kinetics of DSB rejoining was quantified in both scid cells and the parental C.B-17 cells after three different doses of X irradiation: 3, 7.5 and 10 Gy. Repair of DNA DSBs was determined using pulsed-field gel electrophoresis, Southern hybridization and phosphor image analysis. After X irradiation, the cells were allowed to repair at 37 degrees C for up to 1 h or up to 24 h. The most profound difference between the two cell lines was the greatly reduced rate of the slow component of DSB repair in scid cells. C.B-17 cells repaired most of the damage within 1 h, whereas scid cells required 4 to 6 h to reach a similar level after the same dose. No residual or unrepairable DSBs were detected in either cell line 24 h after doses as high as 10 Gy. The scid cells subjected to two doses of 1.5 Gy separated by increasing amounts of time showed no ability to repair sublethal damage between doses, whereas C.B-17 cells receiving two doses of 3.75 Gy separated by increasing periods did show increased levels of survival. These results indicate that scid cells can repair radiation-induced DNA DSBs, although at a reduced rate, but they lack the ability to undergo repair of sublethal damage.

An assay for quantifying DNA double-strand break repair that is suitable for small numbers of unlabeled cells.

A system based on pulsed-field gel electrophoresis (PFGE) is described which measures the induction and repair of DNA double-strand breaks (DSBs) in a biologically relevant X-ray dose range (below 10 Gy) using as few as 125 cells per time. This system was used to measure repair in cells of a freshly obtained human glioblastoma multiforme tumor. No prelabeling of the cells is required, and many different cell types can be studied using this system. Under the pulsed-field conditions used, DNA in the range of 2 to 6 Mb enters the PFGE gel and forms an upper compression zone directly under each well. To quantify the DSBs after electrophoresis, the DNA was transferred to nylon membranes and hybridized with 32P-labeled chromosomal DNA. Phosphor screens were exposed to the membranes and scanned on a phosphor imager. The kinetics of induction and repair was determine by measuring the amount of DNA in the compression zones compared to the amount in the wells. EMT-6 cells were used to demonstrate this method. Induction of DSBs by doses of 0-7.5 Gy X rays was assayed using approximately 12,500 cells per dose and was shown to be linear. Double-strand breaks from 1 Gy were detected above background. To determine a lower limit of the number of cells that could be used to measure DSB repair, cells were embedded in agarose at decreasing concentrations per plug, exposed to 7.5 Gy X irradiation and allowed to repair at 37 degrees C for up to 60 min. DNA from approximately 12,500, 1,250 and 125 cells per time was loaded and subjected to PFGE. The average fast-repair half-time was 3 min and the slow-repair half-time was 35 min. The kinetics of DSB repair in glioblastoma multiforme cells was also determined using this system. Agarose plugs were prepared from a cell suspension, irradiated with 7.5 Gy X rays and allowed to repair for up to 90 min. DNA from approximately 1,250 tumor cells was electrophoresed and analyzed as described above for EMT-6 cells. For this particular tumor, approximately 75% of the induced DSBs were repaired after 90 min. Data presented show that this PFGE-based system is an extremely sensitive method for measuring DSB induction and repair after low doses of X rays using very few cells.

Successful treatment of recalcitrant nonunions with combined magnetic field stimulation.

Nonunions and delayed unions have been classically defined by Bassett as an arrest of the fracture healing process at an intermediary stage of repair, at which time the fracture gap is bridged by fibrocartilage. It is estimated that approximately 10-20 % of long bone fractures in the United States will result in delayed unions when compared to the average rate of healing for the location and type of fracture. Many of these will go on to a nonunion if biological or biomechanical factors are not optimized to enhance healing. Additional commorbities such as smoking, ethanol abuse, malnutrition, malabsorption and altered neurologic conditions can contribute to delayed unions or nonunions. Even despite appropriate and aggressive early management of long bone fractures, a certain percentage still lack progression of healing and go on to nonunion. Classical surgical management of nonunions includes obtaininjg fracture stabilization with ORIF techniques and bone grafting, with reported clinical successes ranging from 50-80%. Those that fail to achieve union despite classical management are indeed recalcitrant nonunions.

Hydroxyurea accelerates the loss of epidermal growth factor receptor genes amplified as double-minute chromosomes in human glioblastoma multiforme.

OBJECTIVE: We sought to determine whether hydroxyurea could accelerate the loss of amplified epidermal growth factor receptor (EGFR) genes from glioblastoma multiforme (GBM). There is good reason to think that elimination of amplified EGFR genes from GBMs will negatively impact tumor growth. Hydroxyurea has previously been shown to induce the loss of amplified genes from extrachromosomal double minutes (dmin) but not from chromosomal homogeneously staining regions. METHODS: Pulsed-field gel electrophoresis and Southern blot hybridization were used to demonstrate EGFR genes amplified as dmin. Giemsa-stained metaphase spreads were prepared in an attempt to visualize dmin. A GBM cell line containing amplified EGFR genes was treated continuously in vitro with 0 to 150 mumol/L hydroxyurea, and slot blot analysis was used to show the loss of amplified EGFR genes. RESULTS: Amplified EGFR genes were found on dmin in 4 of 11 (36%) fresh human GBM biopsy specimens. None of the GBMs contained EGFR genes amplified as homogeneously staining regions. Amplified dmin were not microscopically visible when stained with Giemsa because of their small size. Slot blot analysis showed that these low doses of hydroxyurea accelerated the loss of amplified EGFR genes in a dose- and time-dependent fashion. Pulsed-field gel electrophoresis and Southern blot analysis confirmed that EGFR gene loss was accompanied by amplified dmin loss in a dose-dependent fashion. CONCLUSION: These studies suggest the potential use of low-dose hydroxyurea in the treatment of GBMs.

Pin removal in slipped capital femoral epiphysis: the unsuitability of titanium devices.

In an effort to predict which slipped capital femoral epiphysis fixation devices might cause fewer retrieval problems, 27 consecutive implant-retrieval procedures were reviewed, and mechanical tests using many currently available devices were performed. Problems occurred with both stainless steel and titanium devices if implanted for > 1 year. This tendency for increased problems was statistically significant for the titanium group, and analysis of a retrieved titanium screw showed evidence of osseointegration (direct bone-metal contact at > 90% of the interface) using backscattered image scanning electron microscopic analysis. These clinical data and data from mechanical testing suggest that stainless steel screws or trocar-tipped Steinmann pins are better suited for fixation of slipped capital femoral epiphysis.

Extracorporeal shock-wave lithotripsy in children.

OBJECTIVES: To analyze the efficacy and complication rates of extracorporeal shock-wave lithotripsy in children. METHODS: Between 1987 and 1994, 8760 patients with urinary calculi were treated at our institution. A total of 70 (0.8%) children 3 to 14 years old underwent lithotripsy using the Siemens Lithostar or the Lithostar Plus. A total of 100 calculi in 74 urinary tracts were treated, requiring 129 extracorporeal shock-wave lithotripsy sessions. There were 47 caliceal stones, 31 in the renal pelvis, 16 in the ureter, and 6 staghorn stones. The Lithostar Plus was used in 8 patients, for 3 caliceal, 3 pelvic, and 2 staghorn stones. Follow-up consisted of nephrotomogram or ultrasound 1 day and 1 to 3 months postoperatively. RESULTS: Complete removal of all stone fragments was achieved in 98.5% of the patients after 3 months. Re-treatment was necessary in 20 patients (29.4%). All patients were treated as outpatients, 51 (72.9%) with intravenous sedation and 19 (27.1%) without anesthesia. Complications were present in 7 patients (10%) who had colic and received medical treatment, and convalescence was uneventful. CONCLUSIONS: Extracorporeal shock-wave lithotripsy using the Lithostar and the Lithostar Plus has been demonstrated to be an effective noninvasive procedure to treat radiopaque and even radiolucent or slightly opaque urinary calculi in children.

Induction and repair of DNA double-strand breaks in the same dose range as the shoulder of the survival curve.

We have used pulsed-field gel electrophoresis (PFGE) to test two hypotheses that have been proposed to explain the survival curves with shoulders which are characteristic of low-LET ionizing radiation: (1) Neutral elution studies of the induction of double-strand breaks (DSBs) have suggested that ionizing radiation might induce DSBs in a nonlinear fashion at low doses. (2) Based on analogies to enzyme kinetics, DSB repair might be saturating in the shoulder region. We quantified DSB induction and survival resulting from doses between 0 and 5 Gy spanning the shoulder region of the survival curve. We found that DSB induction was linear at all doses tested down to 0.5 Gy, the limits of sensitivity. Therefore, nonlinear DSB induction cannot account for the shape of the survival curve. To determine whether the DSB repair system was saturated in the shoulder region, we quantified the rate of DSB repair as a function of dose of X rays between 1.25 and 20 Gy. The repair of DSBs was exponential with half-times of repair constant for doses below 10 Gy, and averaged 28 min. We determined the initial rate of repair from the exponential repair kinetics for each dose. The initial rate of repair after radiation treatment increased linearly with dose up to at least 10 Gy. Therefore, saturating DSB repair cannot explain the shoulder of the survival curve.

Hyperthermia inhibits the repair of DNA double-strand breaks induced by ionizing radiation as determined by pulsed-field gel electrophoresis.

Hyperthermia is known to synergistically interact with X-rays to kill cells. We have used pulsed-field gel electrophoresis to investigate the effects of hyperthermia on cell survival and on repair of radiation-induced DNA double-strand breaks (dsbs). Combining hyperthermia (43 degrees C, 45 min) with radiation (7.5 Gy) resulted in a complete inhibition of dsb repair and a surviving fraction of 0.9%. Cells treated with hyperthermia alone resulted in a 55% cell survival with no increase in dsb levels over background. Cells treated with 7.5 Gy alone demonstrated 11% survival and exponential dsb repair. Dsb repair was equally inhibited by hyperthermia whether administered immediately before or after the radiation. We compared the rejoining of dsbs resulting from 7.5 Gy at 37 and 43 degrees C to determine whether dsbs were being repaired during hyperthermia. While repair occurred at 37 degrees C, no dsbs were repaired at 43 degrees C. Our results indicate that hyperthermia completely inhibits dsb repair.

Staining and histomorphometry of microcracks in the human femoral head.

We developed staining techniques that permit identification and histomorphometric analysis of microcracks in the human femoral head 1) from thick, ground bone sections (100 microns) by prestaining with the Villanueva mineralized bone stain (MIBS), and 2) from plastic embedded, undecalcified thin bone sections (5-15 microns) by staining in gallocyanin chrome alum-Villanueva blood stain methods. Both methods represent a significant improvement in the stainability of the microcracks, cellular and tissue elements, and the simultaneous assessment of osteoid seams and tetracycline markers by histomorphometry. Shrinkage and other artifacts were minimized, which helped to clarify some of the uncertainties arising from artifacts resulting from some bone staining methods. Histomorphometric analyses of microcracks were conducted on thick, ground sections of subchondral and trabecular bone. Microcracks were more prevalent in the subchondral bone and osteochondral junction than in the more distant trabeculae. We have consistently localized microcrack areas in bone tissues prepared in these ways.

Measurements of long-term periprosthetic bone changes around a unique composite implant.

Periprosthetic bone changes around a non-bonded composite implant were measured after 6.5 years of implantation. The implanted and unimplanted femora of three canines were cut simultaneously at 3-mm increments. Cortical bone changes at all levels were determined from contact radiographs of the sections using an image analysis system. Alternating sections were cut into 3-mm cubes. The elastic properties of the cancellous bone cubes were determined using ultrasonic techniques. The method of directed secants was used to determine the cancellous bone orientation. The proximal cortical wall thickness on the implanted side increases by an average of 30%. The cancellous bone directly adjacent to the implant was an almost continuous shell encircling the prosthesis. The periprosthetic orientation was circumferential around the implant. The elastic and shear moduli of the cancellous bone increased on the implanted side. The bone changes are currently being correlated with results from finite element calculations.

CpG island mapping of a mouse double-minute chromosome.

The development of double-minute chromosomes (DMs) and subsequent gene amplification are important genomic alterations resulting in increased oncogene expression in a variety of tumors. The molecular mechanisms mediating the development of these acentric extrachromosomal elements have not been completely defined. To elucidate the mechanisms involved in DM formation, we have developed strategies to map amplified circular DM DNA. In this study, we present a long-range restriction map of a 980-kb DM. A cell line cloned from mouse EMT-6 cells was developed by stepwise selection for resistance to methotrexate. This cloned cell line contains multiple copies of the 980-kb DM carrying the dihydrofolate reductase (DHFR) gene. A long-range restriction map was developed in which a hypomethylated CpG-rich region near the DHFR gene served as a landmark. This strategy was combined with plasmid-like analysis of ethidium bromide-stained pulsed-field gels and indicated that a single copy of the DHFR gene was located near a hypomethylated region containing SsII and NotI sites. At least 490 kb of this DM appears to be composed of unrearranged chromosomal DNA.

Induction and repair of DNA double-strand breaks.

Induction and repair of DNA double-strand breaks (DSBs) was measured using a pulsed-field gel electrophoresis system. A cell line of methotrexate-resistant EMT-6 cells that contain numerous double-minutes (DMs) 3 million base pairs in size was employed. The electrophoretic mobility of these DMs depends on whether they have zero, one, or more than one DSB. With no DSBs the DMs remain as circles and are trapped in the origin of electrophoresis, but with one DSB the DMs migrate as a discrete band and can be detected easily through hybridization with a gene-specific probe. Using a clamped homogeneous electrical field apparatus, the induction of DSBs in the 1.5 to 12 Gy X-ray dose range is studied and is shown to be linear. Double-strand break repair following 7.5 Gy is studied, and is shown to be exponential. The kinetics of both induction and repair of DSBs induced in DM DNA was compared to the induction and repair of DSBs in chromosomal DNA and is shown to be similar. The kinetics of repair of DSBs following 7.5 Gy for cells embedded in agarose and cells in suspension is shown to be similar.

Molecular structure and evolution of double-minute chromosomes in methotrexate-resistant cultured mouse cells.

To determine whether microscopically visible double-minute chromosomes (DMs) are derived from submicroscopic precursors, we monitored the amplification of the dihydrofolate reductase (DHFR) gene in 10 independent isolates of methotrexate (MTX)-resistant mouse cells. At every other doubling in MTX concentration, the cells were examined both microscopically, to detect the presence of microscopically visible DMs, and by pulsed-field gel electrophoresis and hybridization to a DHFR-specific probe, to detect submicroscopic DMs. One of the cloned MTX-resistant isolates was examined in detail and was shown to originally contain amplified DHFR genes on circular DMs measuring 1 and 3 Mb in size; additionally, metaphase chromosome preparations from this cloned isolate were examined and were shown to contain microscopically visible DMs too large to enter a pulsed-field gel. During stepwise selection for increasing levels of MTX, the smaller DMs (not microscopically visible) were shown to be preferentially amplified, whereas the larger (microscopically visible) ones decreased in relative numbers. Rare-cutting NotI digestion patterns of total genomic DNA that includes the DMs containing the DHFR gene suggest that the DMs increase in copy number without any further significant rearrangements. We saw no evidence from any of the 10 isolates to suggest that microscopically visible DMs are formed from smaller submicroscopic precursors.

Comparison of ultrasonically applied vs. intra-articular injected hydrocortisone levels in canine knees.

Phonophoresis of hydrocortisone is a commonly performed treatment for a wide variety of soft-tissue as well as intra-articular musculoskeletal disorders. There have been no previously reported studies indicating the degree of hydrocortisone penetration into joints achieved by means of phonophoresis. Twenty-four purebred greyhounds were used to compare intra-articular levels of hydrocortisone resulting from intra-articular injection, phonophoresis, and external application of hydrocortisone. A total of 40 samples from canine knees were analyzed by means of fluorescence polarization. Intra-articular hydrocortisone levels obtained with phonophoresis were extremely low in comparison with those obtained with intra-articular injection. There were no statistically significant differences in intra-articular hydrocortisone levels between the phonophoresis and external application treatments, or between either of these and placebo control measurements taken from knees of untreated dogs and from shoulders of treated dogs. It appears that phonophoresis is an ineffective method of obtaining hydrocortisone penetration into a joint in the canine model.

A canine composite femoral stem. An in vivo study.

To evaluate a carbon fiber/polysulfone composite femoral stem, a press-fit unilateral hemiarthroplasty was performed in 17 greyhounds. The implant was designed to have strength and elastic properties commensurate with the proximal canine femur. The implant geometry was such that the naturally occurring internal cancellous structures of the proximal femur would be preserved and participate in load transfer from the implant to the bone. Animals were killed at one, five, ten, 16, and 24 months. At necropsy all the femoral stems were well fixed and functioning. All implants maintained their structural integrity. Radiographs and computed tomography scans showed a constructive bone remodeling response. Histologic analysis revealed a benign host tissue response, with few inflammatory cells observed. Both bone and fibrous tissue were observed at the implant-host tissue interface. Implants fabricated from carbon/polysulfone composites have the potential for use in load-bearing applications. An implant with appropriate elastic properties provides the opportunity for the natural bone remodeling response to enhance implant stability. Naturally occurring internal cancellous structures can be utilized for load transfer by femoral components. Press-fit devices with no physical or chemical bone-bonding mechanisms can attain long-term successful functional performance.