Expression microarray analysis and oligo array comparative genomic hybridization of acquired gemcitabine resistance in mouse colon reveals selection for chromosomal aberrations

Gemcitabine is a commonly used therapy for many solid tumors. Acquired resistance to this nucleoside analogue, however, diminishes the long-term effectiveness in a majority of patients. To better define the molecular background of gemcitabine resistance, a mouse colon tumor was selected during successive rounds of transplantation with continued treatment of gemcitabine. Expression microarray analysis was applied to determine which genes are consistently and highly overexpressed or underexpressed in the resistant versus the nonresistant tumor. For the statistical interpretation of the microarray data, a parametric model was implemented, which returns model-based differential gene expression (log-) ratios and their uncertainties. This defined a set of 13 genes, putatively responsible for the gemcitabine resistance in solid tumors. One of these, RRM1, was previously identified as an important marker for gemcitabine resistance in human cell lines. Five of the 13 genes, including RRM1, are located within a 3 Mb region at chromosome 7E1 of which four are highly overexpressed, suggesting a chromosomal amplification. Therefore, chromosomal copy number changes were measured, using oligo array comparative genomic hybridization. A narrow and high amplification area was identified on 7E1 that encompassed all five genes. In addition, reduced RNA expression of two other genes at 8E1 encoding COX4I1 and RPL13 could be explained by a decrease in chromosomal copy number on chromosome 8. In conclusion, the array comparative genomic hybridization biologically validates our statistical approach and shows that gemcitabine is capable to select for chromosomally aberrant tumor cells, where changed gene expression levels lead to drug resistance.     

In vivo induction of resistance to gemcitabine results in increased expression of ribonucleotide reductase subunit M1 as the major determinant

Gemcitabine is a deoxycytidine (dCyd) analogue with activity against several solid cancers. Gemcitabine is activated by dCyd kinase (dCK) and interferes, as its triphosphate dFdCTP, with tumor growth through incorporation into DNA. Alternatively, the metabolite gemcitabine diphosphate (dFdCDP) can interfere with DNA synthesis and thus tumor growth through inhibition of ribonucleotide reductase. Gemcitabine can be inactivated by the enzyme dCyd deaminase (dCDA). In most in vitro models, resistance to gemcitabine was associated with a decreased dCK activity. In all these models, resistance was established using continuous exposure to gemcitabine with increasing concentrations; however, these in vitro models have limited clinical relevance. To develop in vivo resistance to gemcitabine, we treated mice bearing a moderately sensitive tumor Colon 26-A (T/C = 0.25) with a clinically relevant schedule (120 mg/kg every 3 days). By repeated transplant of the most resistant tumor and continuation of gemcitabine treatment for >1 year, the completely resistant tumor Colon 26-G (T/C = 0.96) was created. Initial studies focused on resistance mechanisms known from in vitro studies. In Colon 26-G, dCK activity was 1.7-fold decreased; dCDA and DNA polymerase were not changed; and Colon 26-G accumulated 1.5-fold less dFdCTP, 6 hours after a gemcitabine injection, than the parental tumor. Based on in vitro studies, these relative minor changes were considered insufficient to explain the completely resistant phenotype. Therefore, an expression microarray was done with Colon 26-A versus Colon 26-G. Using independently grown nonresistant and resistant tumors, a striking increase in expression of the RRM1 subunit gene was found in Colon 26-G. The expression of RRM1 mRNA was 25-fold increased in the resistant tumor, as measured by real-time PCR, which was confirmed by Western blotting. In contrast, RRM2 mRNA was 2-fold decreased. However, ribonucleotide reductase enzyme activity was only moderately increased in Colon 26-G. In conclusion, this is the first model with in vivo induced resistance to gemcitabine. In contrast to most in vitro studies, dCK activity was not the most important determinant of gemcitabine resistance. Expression microarray identified RRM1 as the gene with the highest increase in expression in the Colon 26-G, which might clarify its complete gemcitabine-resistant phenotype. This study is the first in vivo evidence for a key role for RRM1 in acquired gemcitabine resistance.     

A rehabilitation exercise program to remediate skeletal muscle atrophy in an estrogen-deficient organism may be ineffective

To determine rehabilitation exercise program effects under hormone deficient (ovariectomy or OVX) and hormone supplemented [OVX + 17-beta estradiol (E2)] conditions. Mature female rats (n = 123) were assigned to OVX or OVX + E2-supplemented groups. OVX and OVX + E2 groups were allocated to one of four conditions: (1) control, (2) hindlimb unweighted (HLU) for 4 weeks to induce muscle atrophy, (3) cage Recovery for 2 weeks after HLU, and (4) Recovery with 2 weeks of rehabilitation exercise program after 4 weeks of HLU. Atrophy following HLU was comparable for OVX and OVX + E2-supplemented rats and was significant in all muscles examined (soleus, tibialis anterior, plantaris, gastrocnemius, quadriceps). Also significant with HLU was the decline in muscle force (P < 0.05) in soleus, plantaris, gastrocnemius and tibialis anterior (quadriceps not tested). There were trends toward return of muscle mass in Recovery OVX and Recovery OVX + E2 groups but only the E2 supplemented OVX rats had return of muscle mass (4/5 muscles studied) with exercise. Peak tetanic tension (Po) returned to control values in the E2 supplemented Exercise rats but not in the unsupplemented Exercise group. For example, gastrocnemius Po for OVX HLU, OVX Recovery and OVX-Exercise groups was 82%*, 82%* and 76%* of control. Gastrocnemius Po for E2 supplemented HLU, Recovery and Exercise groups was 72%*, 95% and 106% of control (*P < 0.05 compared to control). H&E cross-sections from OVX-Exercise rats showed central nuclei. In conclusion, a rehabilitation exercise program to remediate acute atrophy in females appears more effective if E2 is present.     

Immune reconstitution in children following chemotherapy for haematological malignancies: a long-term follow-up

Modern intensive chemotherapy for childhood haematological malignancies has led to high cure rates, but has detrimental effects on the immune system. There is little knowledge concerning long-term recovery of the adaptive immune system. Here we studied the long-term reconstitution of the adaptive immune system in 31 children treated for haematological malignancies between July 2000 and October 2006. We performed detailed phenotypical and functional analyses of the various B and T cell subpopulations until 5 years after chemotherapy. We show that recovery of newly-developed transitional B cells and naive B and T cells occurred rapidly, within months, whereas recovery of the different memory B and T cell subpopulations was slower and incomplete, even after 5 years post-chemotherapy. The speed of B and T cell recovery was age-independent, despite a significant contribution of the thymus to T cell recovery. Plasmablast B cell levels remained above normal and immunoglobulin levels normalised within 1 week. Functional T cell responses were normal, even within the first year post-chemotherapy. This study shows that after intensive chemotherapy for haematological malignancies in children, numbers of several memory B and T cell subpopulations were decreased on the long term, while functional T cell responses were not compromised.     

(Pre)malignancies of the female anogenital tract in renal transplant recipients

This mini review describes human papillomavirus-related (pre)malignancies of the lower anogenital tract, for which female renal transplant recipients (RTRs) have a markedly increased risk. Until now, the implementation of intensified cervical cancer screening in RTRs is disappointing. We emphasize the need for improvement of cervical screening programs, combined with close inspection of the vulva and perianal area for potential lethal malignancies in RTRs.     

Pulsed Electromagnetic Fields in the treatment of fresh scaphoid fractures. A multicenter,prospective, double blind,placebo controlled,randomized trial

Background  

The scaphoid bone is the most commonly fractured of the carpal bones. In the Netherlands 90% of all carpal fractures is a fracture of the scaphoid bone. The scaphoid has an essential role in functionality of the wrist, acting as a pivot. Complications in healing can result in poor functional outcome. The scaphoid fracture is a troublesome fracture and failure of treatment can result in avascular necrosis (up to 40%), non-union (5-21%) and early osteo-arthritis (up to 32%) which may seriously impair wrist function. Impaired consolidation of scaphoid fractures results in longer immobilization and more days lost at work with significant psychosocial and financial consequences.     

Dose-finding study of high-dose simvastatin combined with standard chemotherapy in patients with relapsed or refractory myeloma or lymphoma

In vitro statins induce apoptosis in myeloma and lymphoma cells in a dose-and time-dependent way. In combination with dexamethasone and doxorubicin, statins have a chemo-sensitizing effect. Twenty-eight patients with relapsed myeloma or lymphoma were treated with a dose-escalating regimen of simvastatin for 7 days followed by VAD in myeloma patients and CHOP in lymphoma patients. The maximum tolerated dose was 15 mg/kg/day simvastatin. The most frequently reported side-effects were fatigue, gastrointestinal CTC grade 1-2 and neutropenic fever. The dose-limiting toxicity was neutropenic sepsis and grade 3 gastrointestinal side effects. High-dose simvastatin given immediately prior to chemotherapy is safe and tolerable up to a dose of 15 mg/kg/day.     

Influence of oral characteristics and food products on masticatory function

Mastication is a complex process that involves activities of the facial muscles, the elevator and suprahyoidal muscles, and the tongue. These activities result in patterns of rhythmic mandibular movements, food manipulation, and the crushing of food between the teeth. Saliva facilitates mastication by moistening food particles, making a bolus, and assisting swallowing, whereas food consistency modifies masticatory forces, the mandibular jaw movements, the duration of the mastication cycle, and the number of cycles preceding the first swallow. Jaw elevator EMG activity research shows a clear relation between muscular activity and food properties. The teeth, masticatory muscles, and temporomandibular joints are also important because together they form the mechanism by which the food particles are fragmented. Hard and dry foods require more chewing cycles and a longer time in the mouth until swallowing for sufficient breakdown to take place and for enough saliva to be added to form a coherent bolus safe enough for swallowing. Product characteristics, the amount of saliva, dentition, and bite force affect the chewing performance. This study presents an update and synopsis of the effects of saliva, food, dentition, muscle force, and temporomandibular disorders on the masticatory process.