New classification for the treatment of pyogenic spondylodiscitis: validation study on a population of 250 patients with a follow-up of 2 years.

PURPOSE: Pyogenic spondylodiscitis (PS) is still burdened by a high rate of orthopedic and neurological complications. Despite the rising incidence, the choice of a proper orthopedic treatment is often delayed by the lack of clinical data. The aim of this study was to propose a clinical-radiological classification of pyogenic spondylodiscitis to define a standard treatment algorithm. METHODS: Based on data from 250 patients treated from 2008 to 2015, a clinical-radiological classification of pyogenic spondylodiscitis was developed. According to primary classification criteria (bone destruction or segmental instability, epidural abscesses and neurological impairment), three main classes were identified. Subclasses were defined according to secondary criteria. PS without segmental instability or neurological impairment was treated conservatively. When significant bone loss or neurological impairment occurred, surgical stabilization and/or decompression were performed. All patients underwent clinical and radiological 2-year follow-up. RESULTS: Type A PS occurred in 84 patients, while 46 cases were classified as type B and 120 as type C. Average time of hospitalization was 51.94 days and overall healing rate was 92.80%. 140 patients (56.00%) were treated conservatively with average time of immobilization of 218.17 ± 9.89 days. Both VAS and SF-12 scores improved across time points in all classes. Residual chronic back pain occurred in 27 patients (10.80%). Overall observed mortality was 4.80%. CONCLUSIONS: Standardized treatment of PS is highly recommended to ensure patients a good quality of life. The proposed scheme includes all available orthopedic treatments and helps spine surgeons to significantly reduce complications and costs and to avoid overtreatment.

Pregnancy-associated osteoporosis (PAO) with multiple vertebral fragility fractures: diagnosis and treatment in a young primigravid woman.

PAO is an uncommon condition affecting pregnant women during last trimester or early post-delivery period; it is often asymptomatic or presents with pain related to some acute fragility fractures. The diagnosis is often delayed or missed, the etiology remains unknown and no guidelines about treatment have been published. We present one case of PAO in a 33-year-old primigravid woman presenting acute worsening back pain. Our patient was treated with a TLSO brace, oral 25 (OH)-vitamin D supplementation and Teriparatide for 6 months. A short review of the literature has been included and useful advice about how to suspect and diagnose this uncommon disease were given in order to recognize and treat such a debilitating and severe condition for young mothers as best as possible, based on the available scientific evidences.

Kyphoplasty vs conservative treatment: a case-control study in 110 post-menopausal women population. Is kyphoplasty better than conservative treatment?

OBJECTIVE: Osteoporosis is a highly prevalent disease worldwide. Consequences of vertebral osteoporotic fractures include pain and progressive vertebral collapse resulting in spinal kyphosis, decreased quality of life, disability and mortality. Minimally invasive procedures represent an advance to the treatment of osteoporotic VCFs. Despite encouraging results reported by many authors, surgical intervention in an osteoporotic spine is fraught with difficulties. Advanced patients age and comorbidities are of great concern. PATIENTS AND METHODS: We designed a retrospective case-control study on 110 post-menopausal women consecutively visited at our institution. Study population was split in a surgical and a conservative cohort, according to the provided treatment. RESULTS: Kyphoplasty treated patients had lower back pain VAS scores at 1 month as compared with conservatively treated patients (p < 0.05). EQ5D validated questionnaire also showed a better quality of life at 1 month for surgically treated patients (p < 0.05). SF-12 scores showed greater improvements at 1 month and 3 months with statistically significant difference between the two groups just at 3 months (p < 0.05). At 12 months, scores from all scales were not statistically different between the two cohorts, although surgically treated patients showed better trends than conservatively treated patients in pain and quality of life. Kyphoplasty was able to restore more than 54.55% of the original segmental kyphosis, whereas patients in conservative cohort lost 6.67% of the original segmental kyphosis on average. CONCLUSIONS: Kyphoplasty is a modern minimal invasive surgery, allowing faster recovery than bracing treatment. It can avoid the deformity in kyphosis due to VCF. In fact, the risk to develop a new vertebral fracture after the first one is very high.

Clinical predictors of vertebral osteoporotic fractures in post-menopausal women: a cross-sectional analysis.

BACKGROUND: Osteoporosis is a very common bone disorder and accounts for 1.4 million vertebral compression fractures (VCFs) per year, mostly in post-menopausal women. AIM: The aim of this study was to develop a risk scoring system to identify and gauge the risk of osteoporotic VCFs in post-menopausal women. MATERIALS AND METHODS: We conducted a retrospective cross-sectional study on 477 post-menopausal women consecutively visited at our institution. We studied 15 different clinical variables, i.e. age, body mass index (BMI), weight, L1-L4 lumbar T-Score, L1-L4 lumbar Z-Score, L1-L4 lumbar bone mineral density (BMD), femoral neck T-Score, femoral neck Z-Score, femoral neck BMD, smoking habit, alcohol consumption, 25-OH-vitamin D, total alkaline phosphatase, bone alkaline phosphatase, and L4 vertebral volume. Study population was split in a derivation and a validation cohort. A logistic regression model was used to develop a predictive score of osteoporotic VCFs in the derivation cohort, finally the performance of the score was tested in the validation cohort. RESULTS: Age, L1-L4 lumbar T-Score, femoral neck T-Score, L4 vertebral volume, and smoking habit were found to be predictors of VCFs. To each variable a score from 0 to +12 was assigned to the magnitude of regression coefficient. A score ≥ 22 identified VCFs with a sensitivity of 87%/89% and a specificity of 87%/90% in the derivation and validation cohorts, respectively. CONCLUSIONS: Our findings indicate that a simple score derived from clinical history and routine diagnostic workout can be usefully employed to gauge the risk of fragility VCFs in post-menopausal women.

Post-operative spondylodiscitis.

Postoperative spine infections (PSIs) are a frequent and dreaded complication of spine surgery. Although different studies have been published, the prevalence of PSIs is thought to be about 5% for most spine surgical procedures. Different risk factors have been identified for PSIs. Among the others, extensive soft tissue dissection, longer operative time, soft tissue devitalization, and use of surgical instrumentation have been associated with higher risks of infection. Direct inoculation during surgery is the common infection route for PSIs. Gram-positive cocci (such as Staphylococcus aureus, Staphylococcus epidermidis and beta-hemolytic streptococci) are the most common pathogens. Gram-negative bacteria also play a role in PSIs and may be associated with systemic illness and multisystem organ failure. A high level of suspicion is of paramount importance in early diagnosis of PSIs. Clinical symptoms of PSIs may be subtle and the infection may become apparent only in its late stages. Early diagnosis is the most important prognostic factor for PSIs. Although blood tests (i.e. ESR, CRP, and white blood cell count) and imaging studies (most commonly MRI) can be useful, it must be clear to the clinician that diagnostic modalities, either tissue biopsy or blood cultures, are of the utmost importance for diagnosing PSIs and devising a correct antibiotic therapy. Antibiotic therapy with early bracing (or bed rest) is the most commonly used treatment method for PSIs. Nevertheless, a more aggressive surgical treatment may be required in some patients. The goals of surgical treatment are to help the eradication of the infection, provide an adequate wound closure, and maintain spine column mechanical stability.

Medical and surgical treatment of pyogenic spondylodiscitis.

BACKGROUND: Pyogenic vertebral osteomyelitis (PVO) represents approximately 2-7% of all cases of osteomyelitis. The approach to the treatment of PVO may be conservative, which includes antibiotic therapy and orthopaedic treatment, or surgical. AIM: To overview conservative and surigical approaches to PVO. METHODS: A literature review was performed using the Pubmed database to identify studies published in the last 20 years, addressing the treatment of PVO. RESULTS: Empirical antibiotic treatment of PVO, while waiting for the results of cultures or in culture-negative cases, should include broad spectrum agents in association with agents active on Staphylococcus (S.) aureus. Based on local epidemiological data, antibiotics active on methicillin resistant S. aureus (MRSA) should be included. Once an organism has been identified, antibiotics should be initially administered intravenously but the optimal duration of antimicrobial therapy is unclear. Studies have reported that the incidence of treatment failure was higher when i.v. therapy was administered for less than 4 weeks. Rifampin is widely used in the combination therapy of PVO, but no controlled trials are available to define weather this approach is beneficial. Many PVO need a surgical treatment and can represent a real challenge for the orthopaedic surgeon. Anterior and posterior cervical, thoracic, lumbar approaches and the relatives surgical strategies are reported in this review. Moreover, recently the mininvasive posterior stabilization have been proposed as a efficient alternative to open surgery in elderly with severe comorbidities. Possible advantages and limitations of this technique are also reported. CONCLUSIONS: Further research is needed in order to define the optimal duration of antibiotic therapy, and the benefits and limitations of open or mini-invasive surgical techniques.

Surgical treatment of tuberculous spondylodiscitis.

BACKGROUND: Most patients affected by spinal tuberculosis can be successfully treated conservatively with chemotherapy, external bracing and prolonged rest. Nevertheless, kyphotic deformity, spinal instability and neurological deficit remain a common complication associated with conservative approach. AIM: To illustrate different indications and treatment modalities for tuberculous spondylodiscitis, focusing on the role of surgery as an adjuvant of effective chemotherapy in the management of selected patients. MATERIALS AND METHODS: Various early and late surgical procedures are recommended to treat spinal tuberculosis. The Authors analyzed surgical indications, approaches, complications and outcomes comparing their experience with available Literature. RESULTS: Conservative management is preferable in patients without vertebral instability and deformity; in presence of abscesses, invasive radiological techniques in combination with abscess drainage and chemotherapy are recommended. In patients with vertebral collapse, kyphotic deformity or abscess formation, vertebral instability or neurological deficits, anterior radical debridement, anterior strut grafting and anterior instrumentation is an optimal standardized procedure. In patients with involvement of more than two vertebral levels or lumbosacral junction and in those whose sagittal alignment is markedly deformed with segmental kyphosis, and in patients who have difficulty in undergoing anterior instrumentation, posterior instrumentation is recommended in combination with anterior radical debridement and anterior strut grafting in one or two staged procedures. CONCLUSIONS: Since surgery for spinal tuberculosis is demanding, it should be performed only after taking into account the risks and benefits in operable patients. Various surgical procedures are recommended to treat spinal tuberculosis but the common goals are to eradicate the infection and to prevent or to treat neurologic deficits or spinal deformity.

Bioplasty for vertebral fractures: preliminary results of a pre-clinical study on goats using autologous modified skin fibroblasts.

The debate is still ongoing about the long term effects of the mininvasive vertebral augmentation techniques and their usefulness in treating more complex cases where a bone inducing effect more than a merely bone substitution would be suitable, such as the vertebral fractures in young patients. We previously developed a clinically relevant gene therapy approach using modified dermal fibroblasts for inducing bone healing and bone formation in different animal models. The aim of this study is to show the feasibility of a minimally invasive percutaneous intrasomatic ex vivo gene therapy approach to treat thoracolumbar vertebral fractures and anterior column bone defects in a goat model.

Global gene expression of fission yeast in response to cisplatin.

The cellular response to the antitumor drug cisplatin is complex, and resistance is widespread. To gain insights into the global transcriptional response and mechanisms of resistance, we used microarrays to examine the fission yeast cell response to cisplatin. In two isogenic strains with differing drug sensitivity, cisplatin activated a stress response involving glutathione-S-transferase, heat shock, and recombinational repair genes. Genes required for proteasome-mediated protein degradation were up-regulated in the sensitive strain, whereas genes for DNA damage recognition/repair and for mitotic progression were induced in the resistant strain. The response to cisplatin overlaps in part with the responses to cadmium and the DNA-damaging agent methylmethane sulfonate. The different gene groups involved in the cellular response to cisplatin help the cells to tolerate and repair DNA damage and to overcome cell cycle blocks. These findings are discussed with respect to known cisplatin response pathways in human cells.

Protective effect of metallothioneins against oxidative stress evaluated on wild type and MT-null cell lines by means of flow cytometry.

It is generally accepted that metallothioneins (MTs) are devoted to the regulation of the metabolism of essential trace metals and to chelation of toxic metals. Nowadays, there is increasing evidence that MTs also act as free radical scavengers. We employed wild type mouse embryo fibroblast cell line, GKA1, and its MT-null variant, GKA2, in order to correlate the presence of MTs to the sensitivity of cells to reactive oxygen species (ROS), spontaneously generated by the aerobic cellular metabolism, or chemically induced by hydrogen peroxide. The absence of MTs in GKA2 cells was unambiguously correlated to higher sensitivity to ROS attack, as evaluated by detection and quantification of 8-oxo-2'-deoxyguanosine (8-oxo-G), the first product of oxidative attack to DNA, using Fluorescence-Activated Cell Sorter (FACS). When compared to MT-null cell line, the wild type cells (GKA1) were less sensitive to ROS attack. In GKA1 cells, MT biosynthesis is readily induced by Cd2+ treatment, and such an induction caused a further decrease in sensitivity to ROS injury. On the contrary, the MT-null cells (GKA2) expressed no detectable metallothioneins either constitutively, or after heavy metal pretreatment. Indeed, in GKA2 cell line, pretreatment with Cd2+ did not reduce but even enhanced the oxidative stress.

gamma-Glutamyl transpeptidase catalyses the extracellular detoxification of cisplatin in a human cell line derived from the proximal convoluted tubule of the kidney.

Nephrotoxicity is a side-effect and the main factor limiting the clinical use of cisplatin. In vivo, the administration of the cysteine-containing tripeptide glutathione (GSH) has been found to reduce nephrotoxicity, but the biochemical mechanism of this protective action is not fully understood. The present study was designed to gain insights into the mechanism by which GSH prevents cisplatin nephrotoxicity. We also wanted to verify the hypothesis of whether the protective action of GSH is mediated by products of the extracellular breakdown of GSH catalysed by gamma-glutamyl transpeptidase (GGT), an enzyme that is highly expressed in kidney tubular cells. The study was performed in HK-2 cells, derived from the immortalisation of human kidney proximal tubule cells. We investigated the influence of modulators of GGT activity and/or thiols on the antiproliferative activity of cisplatin and on the intracellular GSH content. We determined the antiproliferative activity of cisplatin, platinum cellular accumulation and DNA platination following precomplexing of the drug with thiols. The antiproliferative effect of cisplatin was minimally affected by the addition of GSH. However, when the antiproliferative assay was performed in the presence of glycyl-glycine (GlyGly), to serve as a transpeptidation acceptor and thus to stimulate GGT-mediated GSH catabolism, cisplatin-induced growth inhibition was largely prevented. This effect was not mediated through an increase of intracellular GSH levels, which were not affected by the GlyGly supplementation. The thiol dipeptide cysteinyl-glycine, i.e. the GSH catabolite generated by GGT activity, showed a higher reactivity against cisplatin in vitro than GSH, as was shown by the more rapid oxidation of its -SH groups. The cisplatin/GSH or cisplatin/cysteinyl-glycine adducts did not display an antiproliferative effect. However, 2 h precomplexing with GSH in the presence of GGT, or directly with the GSH catabolite cysteinyl-glycine, decreased the antiproliferative effect of cisplatin and drug-induced DNA platination to a greater extent than precomplexing with GSH alone. The results of the present study show that, in HK-2 cells, extracellular GSH decreases the antiproliferative effects of cisplatin only upon its hydrolysis by GGT, thereby supporting the hypothesis that the extracellular metabolism of GSH by GGT plays a role in modulating cisplatin nephrotoxicity. A primary role in the protection of HK-2 cells appears to be played by cysteinyl-glycine, the proximal product of the GGT-mediated hydrolysis of GSH, which shows a high reactivity against CDDP resulting in the rapid inactivation of the drug.

Effect of metal-based anticancer drugs on wild type and metallothionein null cell lines.

Metallothioneins (MT) are ubiquitous low-molecular-weight metal-binding intracellular proteins. We used wild type mouse embryo fibroblasts, GKA1, and its MT-null variant, named GKA2, in order to correlate the presence of MT to the response to a number of different antitumor drugs with different mechanisms of action. We studied sensitivity of GKA1 and GKA2 cells to metal-based compounds having alkylating property, or able to generate reactive oxygen species (ROS); as well as to drugs acting with different mechanisms. The absence of MT in GKA2 cells was correlated to higher sensitivity to the metal-based drugs compared to that of GKA1. No marked differences in sensitivity of two cell lines against gemcitabine, taxol, and vinblastine were observed. No significant change in sensitivity of either GKA1 or GKA2 cells to these non-alkylating drugs was seen after heavy metal pretreatments. In GKA1 cells, MT biosynthesis was induced by copper and cadmium but not by zinc treatment under the conditions of these experiments. Induction of MT was directly proportional to decrease in sensitivity of GKA1 cells to the compounds used in this experiment. In contrast to GKA1 cells, the MT-null cells (GKA2) expressed no detectable metallothionein either constitutively or after treatment with zinc, copper, or cadmium. Nonetheless, heavy metal pretreatment of GKA2 cells did not cause any change in their sensitivity.

Macrophage stimulating protein (MSP) evokes superoxide anion production by human macrophages of different origin.

1. Macrophage Stimulating Protein (MSP), a serum factor related to Hepatocyte Growth Factor, was originally discovered to stimulate chemotaxis of murine resident peritoneal macrophages. MSP is the ligand for Ron, a member of the Met subfamily of tyrosine kinase receptors. The effects of MSP on human macrophages and the role played in human pathophysiology have long been elusive. 2. We show here that human recombinant MSP (hrMSP) evokes a dose-dependent superoxide anion production in human alveolar and peritoneal macrophages as well as in monocyte-derived macrophages, but not in circulating human monocytes. Consistently, the mature Ron protein is expressed by the MSP responsive cells but not by the unresponsive monocytes. The respiratory burst evoked by hrMSP is quantitatively higher than the one induced by N-formylmethionyl-leucyl-phenylalanine and similar to phorbol myristate acetate-evoked one. 3. To investigate the mechanisms involved in NADPH oxidase activation, leading to superoxide anion production, different signal transduction inhibitors were used. By using the non selective tyrosine kinase inhibitor genistein, the selective c-Src inhibitor PP1, the tyrosine phosphatase inhibitor sodium orthovanadate, the phosphatidylinositol 3-kinase inhibitor wortmannin, the p38 inhibitor SB203580, the MEK inhibitor PD098059, we demonstrate that hrMSP-evoked superoxide production is mediated by tyrosine kinase activity, requires the activation of Src but not of PI 3-kinase. We also show that MAP kinase and p38 signalling pathways are involved. 4. These results clearly indicate that hrMSP induces the respiratory burst in human macrophages but not in monocytes, suggesting for the MSP/Ron complex a role of activator as well as of possible marker for human mature macrophages.

Nitrate leaching beneath a containerized nursery crop receiving trickle or overhead irrigation.

Container production of nursery crops is intensive and a potential source of nitrogen release to the environment. This study was conducted to determine if trickle irrigation could be used by container nursery producers as an alternative to standard overhead irrigation to reduce nitrogen release into the environment. The effect of overhead irrigation and trickle irrigation on leachate nitrate N concentration, flow-weighted nitrate N concentration, leachate volume, and plant growth was investigated using containerized rhododendron (Rhododendron catawbiense Michx. 'Album') supplied with a controlled-release fertilizer and grown outdoors on top of soil-monolith lysimeters. Leachate was collected over two growing seasons and overwinter periods, and natural precipitation was allowed as a component of the system. Precipitation accounted for 69% of the water entering the overhead-irrigated system and 80% of the water entering the trickle-irrigated system. Leachate from fertilized plants exceeded the USEPA limit of 10 mg L(-1) at several times and reached a maximum of 26 mg L(-1) with trickle irrigation. Average annual loss of nitrate N in leachate for fertilized treatments was 51.8 and 60.5 kg ha(-1) for the overhead and trickle treatments, respectively. Average annual flow-weighted concentration of nitrate N in leachate of fertilized plants was 7.2 mg L(-1) for overhead irrigation and 12.7 mg L(-1) for trickle irrigation. Trickle irrigation did not reduce the amount of nitrate N leached from nursery containers when compared with overhead irrigation because precipitation nullified the potential benefits of reduced leaching fractions and irrigation inputs provided under trickle irrigation.

Tachykinin activation of human monocytes from patients with rheumatoid arthritis: in vitro and ex-vivo effects of cyclosporin A.

Three types of tachykinin receptors, namely NK1, NK2 and NK3, are known to preferentially interact with substance P (SP), neurokinin A (NKA) and neurokinin B (NKB), respectively. We previously demonstrated that NK1 and NK2 receptors are present on human monocytes, SP and NKA inducing superoxide anion production and tumor necrosis factor-alpha (TNF-alpha) mRNA expression. NK2 receptor stimulation also triggered an enhanced respiratory burst in monocytes isolated from rheumatoid arthritis (RA) patients. This study was aimed to evaluate the in vitro and ex-vivo effects of cyclosporin A (CsA) on tachykinins-evoked TNF-alpha release from monocytes of healthy donors and RA patients. CsA (100 ng/ml) potently inhibited phorbol ester- and tachykinin-evoked TNF-alpha secretion. In RA patients treated with CsA (Sandimmun Neoral 2.5 mg/kg/day, a significant time-dependent reduction in TNF-alpha secretion from monocytes was measured. This may contribute to the CsA therapeutic activity in RA.

Activity of a trinuclear platinum complex in human ovarian cancer cell lines sensitive and resistant to cisplatin: cytotoxicity and induction and gene-specific repair of DNA lesions.

A collateral sensitivity or a very modest cross-resistance to BBR 3464 was found in 2 ovarian cancer cell lines with experimentally induced resistance to cisplatin. Loss of mismatch repair proteins (hMLH1, hPMS2) or overexpression of nucleotide excision repair proteins (ERCC1) was not detrimental for the cellular sensitivity to BBR 3464. Moreover, interesting differences in the kinetics of formation and removal of DNA lesions at the single-gene (N- ras) level were observed between BBR 3464 and CDDP.

A novel trinuclear platinum complex overcomes cisplatin resistance in an osteosarcoma cell system.

Multinuclear platinum compounds have been designed to circumvent the cellular resistance to conventional platinum-based drugs. In an attempt to examine the cellular basis of the preclinical antitumor efficacy of a novel multinuclear platinum compound (BBR 3464) in the treatment of cisplatin-resistant tumors, we have performed a comparative study of cisplatin and BBR 3464 in a human osteosarcoma cell line (U2-OS) and in an in vitro selected cisplatin-resistant subline (U2-OS/Pt). A marked increase of cytotoxic potency of BBR 3464 in comparison with cisplatin in U2-OS cells and a complete lack of cross-resistance in U2-OS/Pt cells were found. A detailed analysis of the cisplatin-resistant phenotype indicated that it was associated with reduced cisplatin accumulation, reduced interstrand cross-link (ICL) formation and DNA platination, microsatellite instability, and reduced expression of the DNA mismatch repair protein PMS2. Despite BBR 3464 charge and molecular size, in U2-OS and U2-OS/Pt cells, BBR 3464 accumulation and DNA-bound platinum were much higher than those observed for cisplatin. In contrast, the frequency of ICLs after exposure to BBR 3464 was very low. The time course of ICL formation after drug removal revealed a low persistence of these types of DNA lesions induced by BBR 3464, in contrast to an increase of DNA lesions induced by cisplatin, suggesting that components of the DNA repair pathway handle the two types of DNA lesions differently. The cellular response of HCT116 mismatch repair-deficient cells was consistent with a lack of influence of mismatch repair status on BBR 3464 cytotoxicity. Because BBR 3464 produces high levels of lesions different from ICLs, likely including intra-strand cross-links and monoadducts, the ability of the triplatinum complex to overcome cisplatin resistance appears to be related to a different mechanism of DNA interaction (formation of different types of drug-induced DNA lesions) as compared with conventional mononuclear complexes rather than the ability to overcome specific cellular alterations.

The cellular basis of the efficacy of the trinuclear platinum complex BBR 3464 against cisplatin-resistant cells.

Multinuclear platinum compounds have been designed to circumvent the cellular resistance to conventional mononuclear platinum-based drugs. In this study we performed a comparative study of cisplatin and of the triplatinum complex BBR 3464 in a human osteosarcoma cell system (U2-OS) including an in vitro selected cisplatin-resistant subline (U2-OS/Pt). BBR 3464 was extremely potent in comparison with cisplatin in U2-OS cells and completely overcame resistance of U2-OS/Pt cells. In both cell lines, BBR 3464 accumulation and DNA-bound platinum were higher than those observed for cisplatin. On the contrary, a low frequency of interstrand cross-links after exposure to BBR 3464 was found. Differently from the increase of DNA lesions induced by cisplatin, kinetics studies indicated a low persistence of interstrand cross-link formation for BBR 3464. Western blot analysis of DNA mismatch repair proteins revealed a marked decrease of expression of PMS2 in U2-OS/Pt cells, which also exhibited microsatellite instability. Studies on DNA mismatch repair deficient and proficient colon carcinoma cells were consistent with a lack of influence of the DNA mismatch repair status on BBR 3464 cytotoxicity. In conclusion, the cytotoxic potency and the ability of the triplatinum complex to overcome cisplatin resistance appear to be related to a different mechanism of DNA interaction (formation of different types of drug-induced DNA lesions) as compared to conventional mononuclear complexes.

Decreased drug accumulation and increased tolerance to DNA damage in tumor cells with a low level of cisplatin resistance.

In an attempt to examine the cellular changes associated with cisplatin resistance, we selected a cisplatin-resistant (A43 1/Pt) human cervix squamous cell carcinoma cell line following continuous in vitro drug exposure. The resistant subline was characterized by a 2.5-fold degree of resistance. In particular, we investigated the expression of cellular defence systems and other cellular factors probably involved in dealing with cisplatin-induced DNA damage. Resistant cells exhibited decreased platinum accumulation and reduced levels of DNA-bound platinum and interstrand cross-link frequency after short-term drug exposure. Analysis of the effect of cisplatin on cell cycle progression revealed a cisplatin-induced G2M arrest in sensitive and resistant cells. Interestingly, a slowdown in S-phase transit was found in A431/Pt cells. A comparison of the ability of sensitive and resistant cells to repair drug-induced DNA damage suggested that resistant cells were able to tolerate higher levels of cisplatin-induced DNA damage than their parental counterparts. Analysis of the expression of proteins involved in DNA mismatch repair showed a decreased level of MSH2 in resistant cells. Since MSH2 seems to be involved in recognition of drug-induced DNA damage, this change may account for the increased tolerance to DNA damage observed in the resistant subline. In conclusion, the involvement of accumulation defects and the increased tolerance to cisplatin-induced DNA damage in these cisplatin-resistant cells support the notion that multiple changes contribute to confer a low level of cisplatin resistance.