Targets for radiation-induced cell death: when DNA damage doesn't kill.

Chinese hamster ovary (CHO) K1 and radiosensitive CHO irs-20 cells were synchronized in S phase and labeled for 10 min with 5-[(125)I]-iodo-2'-deoxyuridine ((125)IdU). The cells were washed, incubated in fresh medium for 1 h for incorporation of the intracellular radionucleotides into DNA, and then frozen (-80 degrees C) for accumulation of (125)I decays. At intervals after freezing, when the cells had accumulated the desired number of decays, aliquots of the frozen cells were thawed and plated to determine survival. The survival curves for K1 and irs-20 cells were similar from 100% to 30% survival. At higher (125)I doses (more decays/cell), the survival of K1 cells continued to decline exponentially, but the survival of X-ray-sensitive irs-20 cells remained at approximately 30% even after the cells had accumulated 1265 decays/cell. The results contradict the notion that increased DNA damage inevitably causes increased cell death. To account for these findings, we propose a model that postulates the existence of a second radiation target. According to this model, radiation damage to DNA may be necessary to induce cell death, but DNA damage alone is not sufficient to kill cells. We infer from the survival response of irs-20 cells that damage to a second (non-DNA) structure is involved in cell death, and that this structure directly affects the repair of DNA and cell survival.

The transition zone above a lumbosacral fusion.

STUDY DESIGN: The clinical and radiographic effect of a lumbar or lumbosacral fusion was studied in 42 patients who had undergone a posterolateral fusion with an average follow-up of 22.6 years. OBJECTIVE: To examine the long-term effects of posterolateral lumbar or lumbosacral fusion on the cephalad two motion segments (transition zone). SUMMARY OF BACKGROUND DATA: It is commonly held that accelerated degeneration occurs in the motion segments adjacent to a fusion. Most studies are of short-term, anecdotal, uncontrolled reports that pay particular attention only to the first motion segment immediately cephalad to the fusion. METHODS: Forty-two patients who had previously undergone a posterolateral lumbar or lumbosacral fusion underwent radiographic and clinical evaluation. Rate of fusion, range of motion, osteophytes, degenerative spondylolisthesis, retrolisthesis, facet arthrosis, disc ossification, dynamic instability, and disc space height were all studied and statistically compared with an age- and gender-matched control group. The patient's self-reported clinical outcome was also recorded. RESULTS: Degenerative changes occurred at the second level above the fused levels with a frequency equal to those occurring in the first level. There was no statistical difference between the study group and the cohort group in the presence of radiographic changes within the transition zone. In those patients undergoing fusion for degenerative processes, 75% reported a good to excellent outcome, whereas 84% of those undergoing fusion for spondylolysis or spondylolisthesis reported a good to excellent outcome. CONCLUSION: Radiographic changes occur within the transition zone cephalad to a lumbar or lumbosacral fusion. However, these changes are also seen in control subjects who have had no surgery.

Cell cycle-based purging of lymphoma cells from bone marrow harvests using radioiodinated 5-iodo-2'-deoxyuridine.

Minimal residual disease in lymphoma patients is a major problem in the clinical management of their cancer. High-dose chemotherapy followed by autologous bone marrow transplantation has been used to treat the disease. However, residual lymphoma may be reintroduced along with the marrow if it is present in the bone marrow harvest. In this report we describe results of experiments testing the efficacy of 5-[125I]-iodo-2'-deoxyuridine (125IdU) for purging murine RAW117 large cell lymphoma cells (Joshi et al., Oncology 44, 180-185, 1987; Cancer Res. 47, 3551-3557, 1987) from bone marrow in a relevant animal model. Donor BALB/c mice were injected with murine RAW117 cells and euthanized on day 13, and their bone marrow that had been contaminated with tumor cells was harvested and treated in vitro with 125IdU or nonradioactive 127IdU (control). Nine of 10 mice receiving 127IdU-treated bone marrow contaminated with tumor cells died at an average of 17 days after injection. In comparison, 9 of 10 mice injected with 125IdU-treated bone marrow contaminated with tumor cells were still alive after 82 days. In addition, the 125IdU treatment did not diminish the formation of hematopoietic progenitor cell colonies in normal mouse and human peripheral blood stem cells.

Radioiododeoxyuridine in cancer therapy: an in vitro approach to developing in vivo strategies.

UNLABELLED: Studies were undertaken to determine the relationship between IUdR concentration and the duration of radiolabeled IUdR treatment required to incorporate the equivalent of a D(o) dose in vitro and to estimate the treatment parameters necessary to incorporate a killing dose in vivo. METHODS: W138 (normal human) and HeLa (human cancer) cells were grown axenically or in co-culture. The three cultures were treated for 5 days with 18.5 kBq/ml [125I]IUdR. After treatment, the cells were subcultured and grown for 7 days in medium without [125I]IUdR. In separate experiments, Chinese hamster ovary cells (CHO) were labeled with various ratios of radiolabeled (125I) and nonradiolabeled IUdR and the mole rate of IUdR incorporation in double-stranded DNA was measured. Mitotically selected CHO cells were incubated without treatment until > 98% were in S phase. At this time, the cells were labeled for 15 min with several concentrations of either [123I]IUdR or [125I]IUdR and their colony survival was measured. RESULTS: After incubation with [125I]IUdR, selective eradication of HeLa cells from a co-culture of W138 and HeLa cells was achieved. The incorporation of IUdR into DNA of CHO cells, although the sum of a series of enzymatic steps, has the appearance of and can be analyzed as a Michaelis-Menton type curve. The maximum rate of IUdR incorporation (Vmax) is 4.424 x 10(-18) mol/min and the substrate concentration at 1/2 Vmax (K) is 3.717 x 10(-6) M IUdR. The Do dose rates for [123I]IUdR and [125I]IUdR, respectively, are 18.78 and 1.88 initial decays/cell/hr. CONCLUSION: The D(o) dose for *IUdR can be determined from survival curves versus the mole amount of *IUdR incorporated in DNA. To be effective as an in vivo treatment it will be necessary to manipulate the IUdR delivery time, concentration and volume in a manner that assures that the target cells incorporate a cytocidal dose of *IUdR.

The presence of DNA breaks and the formation of chromatid aberrations after incorporation of 125IdUrd may be necessary but are not sufficient to block cell cycle progression in G2 phase.

Cell progression into mitosis and chromatid aberration frequencies were compared in two Chinese hamster ovary (CHO) cell lines after incorporation of 125IdUrd. Asynchronous, exponentially growing populations of CHO K1 and the DNA repair-deficient, radiation-sensitive CHO irs-20 cells were compared after a 10-min exposure to 14.8 kBq/ml 125IdUrd. Essentially no differences were seen for either end point between the cells of the two cell lines. As the cells in S phase at the time of labeling entered the mitotic cell selection window, the number of mitotic cells of each cell line declined to approximately 60% of the respective unlabeled control. Chromosome analysis of the mitotically selected cells indicated an 125I decay-dependent increase in the number of chromatid aberrations in cells of both cell lines. The appearance of aberrations together with the known rates of production and rejoining of DNA double-strand breaks show that cells are able to progress through G2 phase and into mitosis in the presence of such breaks. The data suggest that DNA damage may be necessary, but is not sufficient to cause a radiation-induced blockade of cell progression through G2 phase.

On the safety of 5-[125I]iodo-2'-deoxyuridine--Preclinical evaluation in swine.

To increase tumor incorporation and minimize hepatic degradation of radio-IUdR, compartmental administration routes are being considered as an alternative to intravenous (i.v.) injections. Although there are significant data on the biodistribution and some reports on radiotoxicity of i.v.-administered 125IUdR, similar results for other routes of delivery are not available. We have undertaken a series of experiments intended to examine radiation effects of 125IUdR after intravesical (3 swine; eight 3 mCi doses at 4-day intervals), intracarotid (3 swine; two 10 mCi doses at 2-week intervals), and intra-aortic (5 swine, single dose of 10 mCi) administration in a swine model. Liver, renal functions, and complete blood counts were monitored throughout the duration of the experiment. Pharmacokinetics, systemic distribution of radioactivity and metabolites were measured. The normal tissue 125IUdR uptake and histology were determined after necropsy. No adverse systemic effects were identified. Clinical observations, laboratory data, and necropsy results were within normal range.

The pars defect as a pain source. A histologic study.

STUDY DESIGN: Tissue from the pars defects of six adult patients with symptomatic spondylolysis and spondylolisthesis was obtained at surgery. A histologic study was conducted to identify and characterize neural elements in this tissue. OBJECTIVES: To determine if nociceptive nerve endings were present within the pars defect of patients with symptomatic spondylolysis. SUMMARY OF BACKGROUND DATA: The origin of back pain in patients with spondylolysis remains uncertain. The defect in the pars interarticularis has been implicated as a possible pain source. METHODS: The soft tissue from the pars defect was obtained at surgery. A modified gold chloride stain was used to prepare the tissue for histologic examination. Tissue blocks were sectioned and studied under light microscopy. RESULTS: Neural elements were found in all specimens examined. Free nerve endings believed to have nociceptive function were identified in all specimens. The density of neural elements varied between specimens. CONCLUSIONS: The finding of neural elements, including free nerve endings within the pars defect tissue, suggests that the pars defect may be a source of back pain in some patients with symptomatic spondylolysis.

Do child and/or parent bereavement programs work?

Bereavement programs for children and parents are becoming popular since there are more and more neonatal intensive care programs, pediatric oncology programs and pediatric intensive care programs offering these services. This paper addresses the following question: Do bereavement programs work? An overview of the literature dating back to 1964 was undertaken. Only papers dealing with treatment were reviewed. Randomized controlled trials were selected as presenting the best evidence for or against program effectiveness. Methodologic features such as description of the sample, comparability of treatment and control groups, description of the intervention, use of objective, valid, reproducible and blinded outcome measures, sample size calculations, thoroughness of follow-up and attention to clinical as well as statistical significance were assessed. Four randomized controlled trials were found in the literature search. Two of the studies showed benefit, two did not. All four suffered significant methodologic flaws. At this time it is unclear as to whether or not bereavement programs help families. Given the high costs of health care, it is important that studies be done to determine which families are likely to benefit from bereavement programs and what type of program is effective.

The interosseous membrane of the forearm: structure and its role in Galeazzi fractures.

A cadaveric study was performed to anatomically describe and mechanically document the interosseous membrane of the forearm using gross, histologic, scanning electron microscopic, and mechanical testing. The membrane was found to be a complex structure composed of nerves and vessels but mainly of collagen fibers that thicken to form bands coursing from radius to ulna. Strain-gauge studies demonstrated that the load transfer occurs from the radius to the ulna via the membrane and changes with supination or pronation. With specimens under a specific test load, sectioning of the membrane allowed the fractured radius to shorten by 6.25 mm and sectioning of the triangular fibrocartilage complex resulted in shortening of 7.7 mm. Total shortening after osteotomy and sectioning of the complex structure and membrane ranged from 15 to 40 mm. In Galeazzi fractures, the interosseous membrane acts as a constraint to radial shortening. Anatomic reduction with internal fixation is indicated for this fracture-dislocation.

Blunt injuries to the extracranial cerebral vessels associated with spine fractures.

Injury of the extracranial carotid or vertebral artery with associated spine fractures is a rare but documented entity. In this article, four cases are examined in which patients suffered axial fractures after motor vehicle accidents and subsequently were found to have pathology in one or more of the extracranial arteries. Misdiagnosis is a common complication because symptoms from this are often attributable to closed head injury. Early detection and treatment, however, are essential. As many as 40% of the cases reported have permanent neurologic deficit. Although cerebral angiography remains the diagnostic gold standard, other modalities (eg, transcranial doppler and magnetic resonance angiography) continue to be examined. The treatment of these lesions remains controversial. A variety of surgical procedures may be applicable depending on the time between the injury and the onset of symptoms, the location of the vascular injury, and the rapidity of diagnosis. Anticoagulation therapy appears to play a large role in the management of patients with injury of the extracranial carotid or vertebral artery.

An in vitro 125IUdR-release assay for measuring the kinetics of cell death.

A radionuclide release assay for measuring the in vitro kinetics of cell death has been developed. CHO cells were labelled for 24 h with 3.0 hBq/ml of [125I] iododeoxyuridine (125IUdR) and the fate of the labelled cells and their progeny was monitored at daily intervals by measuring the rate of 125I release. Prelabelling with 125IUdR did not alter the plating efficiency, the doubling time or the selection of mitotic cells. The rate of 125I release from labelled (but otherwise untreated) CHO cells was approximately equal to 4% day. Treatment with a lethal dose of X-rays (30 Gy), heat (46 degrees C, 1 h), cold (-90 degrees C, 1 h) or the antibiotic Geneticin (300 micrograms/ml, continuously) resulted in the release of greater than 99% the 125I activity associated with the cells. Cell death was rapid after heating or freezing, and delayed after treatment with X-rays or Geneticin. The results illustrate the efficacy of the 125I release assay for measuring the kinetics of cell death in mammalian tissue culture cells.

Association between the division delay target and DNA late in the cell cycle.

The precise cell cycle time of association between labeled DNA (the radiation source) and the non-DNA cell structure whose damage is responsible for radiation-induced division delay was measured. Mitotic cells were selected from a monolayer of Chinese hamster ovary cells for 80 min (nine shakes) to establish the rate of cell progression into mitosis. The cell monolayers were then exposed to 0.1295 MBq/ml 125IUdR for 10 min to label the cells in S phase. After pulse labeling, mitotic cell selection was continued for various times (between 0 and 120 min) before 125I decays were accumulated at 4 degrees C. After 2 h in the cold, the cells were rewarmed and the selection of mitotic cells was continued. (Cooling had a small, transient affect on subsequent cell progression.) As the time between labeling and cooling was increased, the fraction of cells selected in mitosis decreased, indicating that an increasing proportion of 125I-labeled cells had entered a sensitive phase of the cell cycle where 125I decays are particularly effective in producing radiation-induced division delay. It is hypothesized that during this sensitive period (from -25 to +90 min of the S/G2 boundary), the labeled DNA comes into sufficiently close contact with a non-DNA structure to facilitate damage to this structure by overlap irradiation from 125I decays in the DNA.

Dynamic EMG analysis of torque transfer in professional baseball pitchers.

Fifteen professional baseball pitchers underwent active pitching motion analysis of the abdominal oblique, rectus abdominis, lumbar paraspinous and gluteus maximus muscles bilaterally via surface electrode evaluation. Baseline resting and isometric maximum values were obtained and active data referenced against these for comparison. The muscle activity then was measured during the pitching sequence and analyzed in each of the five pitching phases. The abdominal oblique, lumbar paraspinous and rectus abdominis contralateral to the pitching arm and the ipsilateral gluteus maximus all had increases in activity level of 75 to 100% during the active pitching motion. Using these data indicating specific muscle group patterns with clinical and performance data, we hope to minimize injuries and maximize pitching performance.

Cell progression after selective irradiation of DNA during the cell cycle.

Chinese hamster ovary cells were labeled with [125I]iododeoxyuridine (125IUdR, 0.1184 MBq/ml for 20 min) and the labeled mitotic cells were collected by selective detachment ("mitotic shake off"). The cells were pooled, plated into replicate flasks, and allowed to progress through the cell cycle. At several times after plating, corresponding to G1, S, late S, and G2 plus M, cells were cooled to stop cell cycle progression and to facilitate accumulation of 125I decays. Evaluation of cell progression into the subsequent mitosis indicated that accumulation of additional 125I decays during G1 or S phase was eight to nine times less effective in inducing progression delay than decays accumulated during G2. The results support our previous hypothesis that DNA damage per se is not responsible for radiation-induced progression delay. Instead, 125I-labeled DNA appears to act as a source of radiation that associates during the G2 phase of the cell cycle with another radiosensitive structure in the cell nucleus, and damage to the latter structure by overlap irradiation is responsible for progression delay (M. H. Schneiderman and K. G. Hofer, Radiat. Res. 84, 462-476 (1980].

Magnetic resonance imaging in the diagnosis of disc degeneration: correlation with discography.

One hundred and one disc levels in 36 patients with low-back pain were studied with magnetic resonance imaging (MRI) (T2-weighted) sagittal images and conventional roentgenographic discography to detect early disc degeneration. Thirty-nine discs also were evaluated after discography with roentgenographic CT MRI findings were compared with discography results. MRI was 99% accurate in predicting normality or abnormality as determined by discography. Changes in disc signal on MRI accurately reflected the presence or absence of degenerative changes seen on discography in patients with low-back pain. Clinically, MRI is a useful technique for detecting early disc degeneration and for assessing the affected disc level and adjacent levels in patients with low-back pain and spondylolithesis.

Evaluation of a new blood autotransfusion device.

A new autotransfusion device was evaluated in dogs. The device uses citrate phosphate dextrose as the blood anticoagulant and automatically delivers the agent in a ratio approximating that found in banked blood. Bleeding, aspiration, and autotransfusion of approximately 3 estimated blood volumes produced small changes in hematologic and coagulation studies. Blood electrolytes stayed within normal ranges. Activated clotting times stayed within normal range after autotransfusion of 2 blood volumes but increased slightly after 3 blood volume transfusions. No significant histopathologic changes were found in any organ system. Rapid infusion of citrated blood causes myocardial depression, which can be reversed by giving calcium. Overall performance of the device was excellent, suggesting further documentation in a clinical setting and evaluation with human blood.