Immunologic function after splenic embolization, is there a difference?

BACKGROUND: To define the immunologic status of patients undergoing splenic embolization (SE) after traumatic injury. This information may lead to the development of immunization protocols based on scientific data. METHODS: Patients with traumatic splenic injury, treated at one level II Trauma Center were eligible for study. SE patients were compared with splenectomy (SP) patients and controls (C = blunt abdominal trauma patients with negative abdominal computed tomography scans). Clinical examination, medical survey, blood sampling, and nuclear medicine spleen scans were performed. IgM, IgG, C3 complement, complement factor B, helper T cells (CD3, CD4), suppressor T-cells (CD8), complete blood counts, and HIV status were tested. Radionuclide spleen scans were analyzed for total spleen volume, splenic defects, abnormal radionuclide uptake, and ectopic sites of tracer uptake. RESULTS: There were no significant differences in age, gender, or injury severity score among groups. Follow-up time was comparable (SP = 2.67 years; SE = 2.88 years). There were no significant differences in all studies measured except for higher CD8 levels in the SP group (730.1 vs. SE 452.1 vs. C 480.6; p = 0.002), although all values were within the normal range. CD3 levels showed a trend of being higher in the SP group (1709.3 vs. SE 1397.2 vs. C 1371.9), but were not statistically significant. CONCLUSION: The data suggest that the immunologic profile of embolized patients is similar to controls. This supports the safe use of SE in managing the traumatically injured spleen. Larger studies examining the immune function after SE will be needed to make definitive vaccination recommendations.

Air medical response to traumatic brain injury: a computer learning algorithm analysis.

BACKGROUND: The role of air medicine in traumatic brain injury (TBI) has been studied extensively using trauma registries but remains unclear. Learning algorithms, such as artificial neural networks (ANN), support vector machines (SVM), and decision trees, can identify relationships between data set variables but are not empirically useful for hypothesis testing. OBJECTIVE: To use ANN, SVM, and decision trees to explore the role of air medicine in TBI. METHODS: Patients with Head Abbreviated Injury Score 3+ were identified from our county trauma registry. Predictive models were generated using ANN, SVM, and decision trees. The three best-performing ANN models were used to calculate differential survival values (actual and predicted outcome) for each patient. In addition, predicted survival values with transport mode artificially input as "air" or "ground" were calculated for each patient to identify those who benefit from air transport. For SVM analysis, chi was used to compare the ratio of unexpected survivors to unexpected deaths for air- and ground-transported patients. Finally, decision tree analysis was used to explore the indications for various transport modes in optimized survival algorithms. RESULTS: A total of 11,961 patients were included. All three learning algorithms predicted a survival benefit with air transport across all patients, especially those with higher Head Abbreviated Injury Score or Injury Severity Score values, lower Glasgow Coma Scale scores, or hypotension. CONCLUSION: Air medical response in TBI seems to confer a survival advantage, especially in more critically injured patients.

Asphyxia: a rare cause of death for motor vehicle crash occupants.

Motor vehicle related trauma is one of the leading causes of traumatic death. Although most of these deaths are because of severe blunt force trauma, there are people without severe injury who die of asphyxia related to the motor vehicle collision. There were 37 deaths because of motor vehicle related asphyxia in San Diego County during 1995-2004. Almost half (48.6%) of these deaths were because of compression asphyxia, 29.7% were positional asphyxia deaths, and 16.2% died of a combination of compression and positional asphyxia. We were unable to classify the mechanism of asphyxia for the remaining 5.4% of asphyxia deaths. Almost all occupants dying from asphyxia were involved in rollover crashes and may have been incapacitated by obesity, drug or alcohol intoxication, or blunt force trauma. Compression asphyxia deaths occurred both from vehicle crush with intrusion into the passenger compartment and from ejection of the occupant and subsequent crushing by the vehicle. Positional asphyxia occurred in positions interfering with normal respiration, including inversion. None of the occupants had injury severe enough to result in death at the scene if they had not first died of asphyxia. This study suggests classifying the mechanism of asphyxia for these fatalities may be a challenge to forensic pathologists who seldom see these rare deaths.

Acquired cisplatin resistance in human ovarian cancer cells is associated with enhanced repair of cisplatin-DNA lesions and reduced drug accumulation.

Studies were undertaken to investigate acquired resistance to cisplatin in human ovarian cancer cells. The cell lines A2780 and A2780/CP70 were studied to assess their respective characteristics of drug accumulation and efflux, cytosolic inactivation of drug, and DNA repair. All experiments were performed using 1-h drug exposures. The A2780/CP70 cell line was 13-fold more resistant to cisplatin than A2780 cells. When studied at their respective IC50 doses, drug accumulation rates were similar for the two cell lines. However, the resistant cell line was twofold more efficient at effluxing drug, which was associated with reduced total drug accumulation for equivalent micromolar drug exposures. At equivalent levels of total cellular drug accumulation, the two cell lines formed the same levels of cisplatin-DNA damage, suggesting that cytosolic inactivation of drug does not contribute to the differential in resistance between these cell lines. Resistant cells were also twofold more efficient at repairing cisplatin-DNA lesions in cellular DNA and in transfected plasmid DNA. We conclude that in these paired cell lines, alterations in drug uptake/efflux and in DNA repair are the major contributing factors to acquired resistance to cisplatin.

Modulation of cisplatin sensitivity and growth rate of an ovarian carcinoma cell line by bombesin and tumor necrosis factor-alpha.

A twofold change in the cisplatin (DDP) sensitivity of 2008 human ovarian carcinoma cells is sufficient to reduce tumor response in vivo. The DDP sensitivity of these cells can be enhanced by activation of the epidermal growth factor and protein kinase C signal transduction pathways. We report here that two endogenous growth factors, bombesin and tumor necrosis factor alpha (TNF alpha), enhanced DDP sensitivity by factors of 1.7 +/- 0.1 (SD)-fold and 1.8 +/- 0.1 (SD)-fold, respectively. Both agents also produced sensitization in an 11-fold DDP-resistant 2008 subline. Neither bombesin nor TNF alpha changed the accumulation of DDP, glutathione content, or glutathione-S-transferase activity in 2008 cells. However, a 2-h exposure to both bombesin and TNF alpha was sufficient to increase 2008 cloning efficiency by up to 2.6 +/- 0.1 (SD)-fold and 2.2 +/- 0.1 (SD)-fold, and it increased average colony size by 1.35 +/- 0.1 (SD)-fold and 1.55 +/- 0.1 (SD)-fold, respectively. Bombesin increased intracellular free calcium, and this was blocked by the bombesin receptor-specific antagonist SC196, demonstrating that 2008 cells have functional bombesin receptors. These results indicate that bombesin and TNF alpha can enhance sensitivity to DDP in both DDP sensitive and resistant variants of a human ovarian carcinoma and that both agents serve as growth factors for this tumor.

DNA-mediated transfer and expression of a human DNA repair gene that demethylates O6-methylguanine.

Human liver DNA was transfected into CHO cells (mex-) along with pSV2gpt and colonies were selected first for resistance to mycophenolic acid and then to chloroethylnitrosourea. Transformants were obtained that contained approximately 10,000 molecules of O6-alkylguanine alkyltransferase (mex+) per cell. Their genome contained at least three copies of the human Alu sequence.

The impact of safety belt use on liver injuries in motor vehicle crashes: the importance of motor vehicle safety systems.

BACKGROUND: Liver injuries (LIs) are one of the most serious and common consequences of motor vehicle crashes (MVCs). In the unstable patient, early detection of LI based on clinical suspicion will improve acute trauma care and outcomes. The specific objectives of this analysis are to identify crash scene and occupant risk factors for LI from MVC. METHODS: Crash Injury Research and Engineering Network data were used to identify risk factors for LI; age, sex, safety belt use, air bag deployment, DeltaV (change in velocity), principal direction of force, vehicle crush, and intrusion. Occupants with LI were compared with four control groups without LI; (1) no abdominal (ABD) injury (NO_ABD), (2) any ABD (ANY_ABD), (3) ABD Abbreviated Injury Scale score of 1 to 2 (ABD_1-2), and (4) ABD Abbreviated Injury Scale score of 3 or more (ABD_3+). LI occupants were compared with each control group and odds ratios (OR) for risk of LI were computed. RESULTS: There were 311 Crash Injury Research and Engineering Network subjects aged 5 or more years with LI. The total mean Injury Severity Score was 37.6. LI was strongly and significantly associated with safety belt restraint use without air bag deployment, compared with each control group: Liver injury - restrained + air bag not deployed versus (1) NO_ABD, N = 1,519; OR = 4.4, (2) ANY_ABD, N = 317; OR = 2.6, (3) ABD_1 to 2, N = 155; OR = 3.1, (4) ABD_3+, N = 217; OR = 2.4 (p < 0.001). This association was independent of driver or passenger status and principal direction of force. LIs were also strongly and significantly associated with greater vehicle interior intrusion. CONCLUSIONS: LIs were strongly associated with a safety belt restraint in use in the absence of air bag deployment during MVC. This data may have profound importance to the trauma surgeon as an early indicator for LI during resuscitation. These findings also have important implications for future research efforts to improve safety systems in motor vehicles and reduce morbidity and mortality from MVCs in the United States.

Motor vehicle-related cardiac and aortic injuries differ from other thoracic injuries.

BACKGROUND: Traumatic cardiac and thoracic aortic injuries are hypothesized to result from rapid deceleration of occupants during a motor vehicle crash. The purpose of this study was to identify potential risk factors for motor vehicle-related cardiac and thoracic aortic (HTA) injury using the Crash Injury Research Engineering Network (CIREN) database. METHODS: CIREN data were used to test the hypothesis that there is no difference between occupants with HTA injury and occupants with thoracic injury other than the heart or aorta (non-HTA). Occupant variables (restraint use, airbag deployment, Glasgow Coma Scale score, Injury Severity Score, concomitant injuries, driver versus passenger status, height, and comorbidity) and crash variables (principal direction of force, change in velocity, vehicle crush, intrusion, and vehicle type) were compared for these two groups. Odds ratios were used to quantify the potential risk factors for HTA injury compared with non-HTA injury. RESULTS: There were 168 occupants with an HTA injury and 731 with a non-HTA injury. Greater crash severity (based on vehicle crush and change in velocity), improper safety belt use, and lack of safety belt use were significantly associated with HTA injury. Unrestrained occupants had almost three times the chance of having an HTA injury (odds ratio = 2.86; p < 0.05). For restrained drivers, 41.4% of HTA injuries were caused by vehicle interior components. When not protected by both safety belts and air bags, 45.7% of driver HTA injuries were caused by the steering wheel. For passengers, the vehicle interior (armrests, side interior, and B-pillars) accounted for most HTA injuries regardless of safety system status. More than half of all occupants wearing safety belts who sustained an HTA injury were improperly wearing their safety belts. CONCLUSION: The high mortality associated with cardiac and aortic injuries supports the need to prevent these injuries from occurring during motor vehicle crashes. These results suggest proper use of safety belts is necessary to prevent cardiac and thoracic aortic injuries. However, other important potential risk factors, such as motor vehicle size and crash severity, might continue to present a challenge to motor vehicle safety professionals.

Calcaneal fractures in occupants involved in severe frontal motor vehicle crashes.

The calcaneous is the largest tarsal bone in the foot and plays an important role in walking and running. Motor vehicle crashes and falls from elevation have been associated with calcaneal fractures. Although not life-threatening, these injuries may result in permanent disability. This study used the Crash Injury Research and Engineering Network (CIREN) database to describe calcaneal fractures and concomitant lower extremity skeletal injury patterns for occupants involved in motor vehicle crashes. Sixty-three drivers and 7 front row passengers with calcaneal fractures were identified in the CIREN database during 1997-2005. Almost all these occupants were involved in severe (based on the delta V and vehicle crush) frontal or off-set frontal crashes with toe pan intrusion. Eighty-four percent of the calcaneal fractures were intra-articular or partially articular. Overall, 93% of occupants also had injury to other body regions with 84% having other lower extremity fractures. One year after the crash, most occupants had not returned to their prior level of physical functioning. Surgically managing patients with calcaneal fractures for an optimal outcome remains a challenge for orthopedic surgeons. Because lower extremity injuries, including calcaneal fractures, may cause permanent disability, it is important to prevent these injuries through structural improvements in motor vehicle design.

Fatal positional asphyxia associated with rollover crashes.

Motor vehicle rollover crashes result in complex occupant kinematics with the potential for severe injury. Five cases of fatal asphyxia in occupants suspended from their safety belt upside down after a rollover crash are presented. These fatalities accounted for 13.5% of all motor vehicle related asphyxia deaths in San Diego County over a 10-year period. This study supports previous research noting that incapacitation due to other injuries, alcohol, or obesity may be associated with fatal positional asphyxia due to inversion during rollovers. Safety belts are proven to prevent serious injury in motor vehicle crashes and should always be worn. However, redesign of the buckle could be considered to permit easier release by an occupant. We also suggest that pre-existing heart disease may contribute to the possibility of a fatal asphyxia outcome. Although this is a rare cause of motor vehicle related death, our results suggest that these are potentially preventable deaths.

Traumatic brain injury outcomes in pre- and post- menopausal females versus age-matched males.

Gender differences in outcomes from major trauma have been described previously, and exogenous female hormone administration appears to be neuroprotective following traumatic brain injury (TBI). This analysis explored outcomes in pre- and post-menopausal females versus age-matched males. A total of 13,437 patients (n = 3,178 females, n = 10,259 males) with moderate-to-severe TBI (head AIS > or = 3) were identified from our county trauma registry. Overall mortality was similar between males and females (22% for both). Logistic regression was used to compare gender outcome differences, with a separate analysis performed for premenopausal (< 50 years) versus postmenopausal (> or = 50 years) patients, and after stratification by decade of life. No statistically significant difference in outcomes was observed for pre-menopausal females versus males (odds ratio [OR] 1.06; 95% confidence interval [CI] 0.83, 1.35; p = 0.633), but outcomes were significantly better in postmenopausal females versus males (OR 0.63, 95% CI 0.48-0.81, p < 0.001) after adjusting for age, mechanism of injury, Glasgow Coma Scale (GCS), hypotension (SBP < or = 90 mm Hg), head Abbreviated Injury Score (AIS), and Injury Severity Score (ISS). Stratification by decade of life revealed the gender survival differential inflection point to occur between ages 40-49 (OR 1.06, 95% CI 0.66-1.71, p = 0.798) and ages 50-59 (OR 0.38, 95% CI 0.20-0.74, p = 0.005). In addition, Revised Trauma Score and Injury Severity Score (TRISS) was used to calculate probability of survival (PS); all patients were then stratified by decade of life, and males and females were compared with regard to mean survival differential (outcome - PS). The identical pattern of improved outcomes in post-menopausal but not pre-menopausal females versus age-matched males was observed. These data suggest that endogenous female sex hormone production is not neuroprotective.

Rollover crashes: predicting serious injury based on occupant, vehicle, and crash characteristics.

The purpose of this research was to determine occupant, vehicle, and crash characteristics predicting serious injury during rollover crashes. We compared 27 case occupants with serious or greater severity injuries with 606 control occupants without injury or with only minor or moderate injury. Odds ratios (OR) for individual variables and logistic regression were used to identify predictive variables for serious injury associated with rollovers. Cases more often had thorax, spine, or head injury compared to controls that more often had extremity injuries. Intrusion (especially roof rail or B-pillar intrusion) at the occupant's position, the vehicle interior side and roof as sources of injury, and improper safety belt use were significantly associated with serious injury. Even when safety belt use or proper use was controlled for, occupants with greater magnitude of intrusion at their seat position were about 10 times more likely to receive serious injury. Although prevention of rollover crashes is the ultimate goal, it is important to develop safer vehicles and safety systems to better protect occupants who are involved in rollover crashes. This also requires improvement in data collection systems documenting these types of crashes.

Analysis of events associated with cell cycle arrest at G2 phase and cell death induced by cisplatin.

DNA is the accepted target for cisplatin, but recent evidence has shed doubt on DNA synthesis as the critical process. L1210/0 cells incubated for 2 hours with cisplatin progress to the G2 phase of the cell cycle and are arrested there for several days. They then either progress in the cell cycle or die. In cells that eventually die, total transcription, polyadenylated [poly(A)+] RNA synthesis, and protein synthesis were markedly inhibited only after 48 hours. Nicotinamide adenine dinucleotide (NAD) and adenosine triphosphate (ATP) levels decreased after 3 days. Cell membrane integrity was lost after 4 days. These results demonstrate that cells can be lethally damaged, yet continue to undergo apparently normal metabolic activities for several days. In a previous study, DNA double-strand breaks were detected after 1 day. We now show that by 2 days, breaks are visible as fragmentation in the nucleosome spacer regions of chromatin. This type of damage is consistent with cell death occurring by the process of apoptosis. Cell shrinkage and morphology were also consistent with this type of cell death. The slow cell death reported here appears to occur at the G2/M transition and may involve events that normally occur at this stage of the cell cycle. These results demonstrate the importance of DNA degradation as an early and possibly essential step in cell death.

UCN-01: a potent abrogator of G2 checkpoint function in cancer cells with disrupted p53.

BACKGROUND: Arrest of the cell cycle in G2 phase following DNA damage helps protect cell viability by allowing time for DNA repair before entry into mitosis (M phase). Abrogation of G2 arrest sensitizes cells to the effects of DNA-damaging agents. UCN-01 (7-hydroxystaurosporine), a protein kinase C inhibitor that may block G2 checkpoint regulation, has been reported to enhance the cytotoxicity of mitomycin C, a known DNA-damaging agent. PURPOSE: We studied the effect of UCN-01 on G2 checkpoint control in human lymphoma CA46 cells, whose sensitivity to various DNA-damaging agents and G2 response to DNA damage have been characterized. We also assessed the ability of UCN-01 to enhance the cytotoxicity of gamma irradiation in CA46 cells and human colon carcinoma HT-29 cells, both of which are mutant for p53 function. The influence of p53 function on UCN-01-mediated abrogation of the G2 checkpoint and enhancement of DNA-damaging agent cytotoxicity was studied in transfected human breast carcinoma MCF-7 cells that either expressed or did not express the human papillomavirus type-16 E6 protein. MCF-7 cells have normal p53 function, and the E6 protein binds p53 protein and promotes its destruction. METHODS: The effect of UCN-01 on cell cycle arrest induced by gamma irradiation was studied in CA46 cells and in transfected MCF-7 cells by use of flow cytometry. A histone H1 phosphorylation assay was employed to measure cyclin B1/Cdc2 kinase activity in extracts derived from irradiated and nonirradiated CA46 cells that had been either treated or not treated with UCN-01; the phosphorylation status of Cdc2 kinase protein in the same extracts was determined by use of western blotting. The effect of UCN-01 on the cytotoxicity of gamma irradiation in CA46 and HT-29 cells was determined by use of MTT (thiazolyl blue) and clonogenic (colony-forming) assays, respectively; a clonogenic assay was also used to measure the effect of UCN-01 on the cytotoxicity of cisplatin in transfected and nontransfected MCF-7 cells. RESULTS: G2 arrest induced in CA46 cells by gamma irradiation was minibited by treatment with UCN-01 in a dose-dependent manner; arrest in G2 was completely abrogated by exposure to 300 nM UCN-01. Biochemical markers indicative of the G2/M transition, including the activation of cyclin B1/Cdc2 kinase and the suppression of Cdc2 threonine-14 and tyrosine-15 phosphorylation, were detected in irradiated cells treated with UCN-01. UCN-01 enhanced the cytotoxicity of gamma irradiation in CA46 and HT-29 cells. MCF-7 cells with functional p53 protein were more resistant to G2 checkpoint abrogation by UCN-01 than MCF-7 cells with disrupted p53 function. UCN-01 markedly enhanced the cell-killing activity of cisplatin in MCF-7 cells defective for p53 function. CONCLUSIONS AND IMPLICATIONS: UCN-01 is a potent abrogator of G2 checkpoint control in cancer cells with disrupted p53 function. UCN-01 might be capable of enhancing the effectiveness of DNA-damaging agents in the treatment of tumors with cells lacking normal p53 function.

Persistent binding of 3-methylcholanthrene to mouse lung DNA and its correlation with susceptibility to pulmonary neoplasia.

A/J, C3H/HeJ, DBA/2J, and C57BL/6J mice have different susceptibilities to polycyclic aromatic hydrocarbon-induced pulmonary neoplasia, whereas the livers from these animals are uniformly resistant to the carcinogenic actions of these substances. After i.v. injection of [3H]-3-methylcholanthrene, radiolabel was detected in the DNA of both lung and liver of all these strains. The DNA was digested to deoxyribonucleosides and chromatographed on Sephadex LH-20. The amounts of nucleoside-bound adducts detected varied markedly with the different tissues and strains. These adducts were undetectable in liver DNA by 28 days. Although all lung preparations showed some reduction in adducts by 28 days, the amounts in A/J lung were always highest; this correlated with its high susceptibility to neoplastic transformation. In all preparations, radioactivity eluted from Sephadex LH-20 with the column void volume or with underivatized nucleosides. Tissue, but not strain differences were observed in these chromatographic profiles. The predominance of these early-eluting peaks in liver, rather than lung, suggests that they may represent noncarcinogenic lesions. This radiolabeled material remains uncharacterized, but some possibilities are discussed.

Mechanism of cis-diamminedichloroplatinum(II)-induced cytotoxicity: role of G2 arrest and DNA double-strand breaks.

DNA has been implicated as the critical target for cis-diamminedichloroplatinum(II) (cis-DDP)-induced cytotoxicity. In vitro, DNA-platinum adducts inhibit DNA synthesis. An assessment of the inhibition of DNA synthesis in murine leukemia L1210 cells demonstrated that, although cell division was halted, DNA replication continued for a period of time. The DNA underwent almost a complete doubling even in cells that did not divide. Flow cytometric analysis demonstrated a slowed synthetic phase which progressed to a block in the G2 phase of the cell cycle. The duration of the G2 block was proportional to the concentration of cis-DDP. Low concentrations of cis-DDP caused the cells to be transiently blocked in the G2 phase for 24 to 48 h. Higher concentrations of cis-DDP resulted in a G2 arrest that was not reversed by 96 h. After this time, the arrested cells appeared to disintegrate, rather than recover. Cell survival and trypan blue exclusion studies indicated that, at low drug concentrations, cells which had transiently arrested in the G2 phase survived, while at higher concentrations only a limited number of survivors were responsible for the observed recovery of growth. Analysis of DNA double-strand breaks showed that significant numbers of breaks only occurred at concentrations of cis-DDP that subsequently led to debris detectable on the flow cytometer and to loss of trypan blue exclusion. The formation of these breaks appeared to be the first detectable change that was indicative of cell death. It is proposed that cells arrest in the G2 phase because they are unable to transcribe damaged DNA and make mRNA essential for passage into mitosis. DNA repair probably overcomes this arrest. Cell death may therefore be a consequence of the inability to adequately recover transcription.

Influence of cis-diamminedichloroplatinum(II) on DNA synthesis and cell cycle progression in excision repair proficient and deficient Chinese hamster ovary cells.

DNA has been implicated as the critical intracellular target for cis-diamminedichloroplatinum(II) (cis-DDP) action. Inhibition of DNA synthesis is a consequence of platination and has become accepted as the critical step in cis-DDP-induced toxicity. We have previously demonstrated that, following incubation with cis-DDP, murine leukemia L1210 cells progress through synthesis only to arrest in the G2 phase of the cell cycle. The G2 arrest was transient at low drug concentrations and was persistent at higher concentrations with a concomitant loss of viability. Chinese hamster ovary cell lines both proficient and deficient for DNA excision repair have been used to analyze the relationship between inhibition of DNA synthesis and toxicity and to determine whether DNA repair is necessary for cell cycle progression. Two repair-deficient cell lines were hypersensitive to cis-DDP and demonstrated a marked arrest in the G2 phase. The arrest was transient over only a small range of concentrations. At higher concentrations, the arrest was persistent and the cells subsequently died. Incorporation of [3H]thymidine into macromolecules demonstrated no inhibition of DNA synthesis while these cells progressed through the S phase. In contrast, at higher, but nontoxic, concentrations of cis-DDP, the repair-proficient cells exhibited inhibition of DNA synthesis while in S. At toxic concentrations, these cells also arrested in G2. Therefore, direct inhibition of DNA synthesis correlated only with the concentration of drug and not with the different sensitivities of the cell lines. Arrest of cells in G2 did correlate with toxicity. In every cell line, the appearance of G2-arrested cells preceded cell disintegration. It is proposed that the G2-arrested cells preceded cell disintegration. It is proposed that the G2 arrest results from the inability of the cells to transcribe genes required for passage into mitosis. Cells proficient in DNA repair can circumvent this arrest by repairing the damaged DNA and permitting transcription to proceed. These results support the hypothesis that inhibition of DNA synthesis is not the critical step in cis-DDP-induced cytotoxicity.

Multiple mechanisms of resistance to cis-diamminedichloroplatinum(II) in murine leukemia L1210 cells.

As an experimental model for resistance to cis-diamminedichloroplatinum(II) (cis-DDP), murine leukemia L1210 cells have been exposed to a stepwise increase in cis-DDP concentration to produce a variety of resistant cell lines. Intraspecies hybrids of the sensitive and resistant cells were made to determine whether cis-DDP resistance is a dominant or recessive trait. Hybrid cells displayed a partial degree of resistance as compared to the parental cells. To determine whether this was due to a single codominant trait or contribution from a variety of resistance mechanisms, the cells and hybrids were investigated for alterations in the accumulation of drug, as well as alterations in glutathione levels which might inactivate the drug. The cis-DDP-resistant cells demonstrated both a 50% reduction in accumulation of drug and a 1.7-fold increase in intracellular glutathione. Reducing the glutathione levels in these cells with buthionine sulfoximine did not sensitize them to cis-DDP. The hybrid cells had the same accumulation and the same levels of glutathione as the cis-DDP-sensitive cells. Parallel studies were performed with cells resistant to 1,2-diaminocyclohexaneplatinum(II) analogues. These cells also demonstrated reduced drug accumulation but no increase in glutathione. Therefore, both a decrease in accumulation and increase in glutathione may mediate resistance. Both mechanisms represent recessive traits as demonstrated in the cell hybrids. These mechanisms can only account for a small part of the resistance in these cells. A major, dominant mechanism occurs after the DNA has been platinated, but it remains to be determined whether this involves DNA repair, postreplication repair, or some other as yet unidentified process.

Irradiation-induced expression of O6-methylguanine-DNA methyltransferase in mammalian cells.

O6-Methylguanine-DNA methyltransferase (MGMT) is a DNA repair protein which plays an important role in chemotherapy, mutagenesis, and carcinogenesis. The specific activity of MGMT in female rat liver can be induced by approximately 20-fold by treatment of the rats with gamma-irradiation. Maximum response occurred 48 h after 15 Gy irradiation. MGMT levels in male rats were induced by only 3-fold. MGMT activity was also induced by irradiation of rat hepatoma H4IIE cells with a 3-fold increase noted after treatment with 3 Gy. Northern analysis and nuclear run-on assays indicated that the induction of MGMT was regulated at the transcriptional level. The radiation-mediated increase in MGMT was blocked by H7, a protein kinase inhibitor, but not by H89, an inhibitor of protein kinase A. Hydroxyl radicals may play a role in the induction mechanism since dimethyl sulfoxide, a radical scavenger, blocked the radiation-mediated increase in MGMT. MGMT activity was also increased by treatment of the cells with H2O2, in accordance with the involvement of activated oxygen species in the induction of MGMT. Finally, the addition of cycloheximide, an inhibitor of protein synthesis, prior to but not after irradiation, abolished the increase in MGMT activity.