Programmed cellular response to ionizing radiation damage

Typically, economic evaluation compares the costs and benefits of two or more interventions and seeks to identify the single superior option on the basis of relative cost-effectiveness. It is then anticipated that all patients will receive the more or most cost-effective option. This 'all or nothing' approach can be departed from when sub-groups of patient exist, defined on the basis of clinical or demographic characteristics which are considered to influence benefit, for whom an option is cost-effective whilst not being so for the population of patients as a whole. However, patients' preferences concerning the different process characteristics and outcomes of an intervention will also influence the benefit they derive from health care. This paper explores the concept of preference-based sub-group analysis in economic evaluation to assess the potential cost-effectiveness of using patients' preferences to determine treatment allocation. The clinical example used to explore these methods is the comparison of abdominal hysterectomy (AH) and transcervical resection of the endometrial (TCRE) for the treatment of menorrhagia.     

Role of ceramide in cellular senescence

Recently the sphingomyelin cycle, involving the hydrolysis of membrane sphingomyelin by an activated sphingomyelinase to generate ceramide, has emerged as a key pathway in cell differentiation and apoptosis in leukemic and other cell types. Here we investigate a role for this pathway in the senescence of WI-38 human diploid fibroblasts (HDF). We found that endogenous levels of ceramide increased considerably (4-fold) and specifically (compared with other lipids) as cells entered the senescent phase. Investigation of the mechanism of increased ceramide led to the discovery that neutral sphingomyelinase activity is elevated 8-10 fold in senescent cells. There were no changes in sphingomyelinase activity or ceramide levels as HDF entered quiescence following serum withdrawal or contact inhibition. Thus, the activation of the sphingomyelinase/ceramide pathway in HDF is due to senescence and supports the hypotheses that senescence represents a distinct program of cell development that can be differentiated from quiescence. Additional studies disclosed the ability of ceramide to induce a senescent phenotype. Thus, when exogenous ceramide (15 microM) was administered to young WI-38 HDF, it produced endogenous levels comparable to those observed in senescent cells (as determined by metabolic labeling studies). Ceramide concentrations of 10-15 microM inhibited the growth of young HDF and induced a senescent phenotype by its ability to inhibit DNA synthesis and mitogenesis. These concentrations of ceramide also induced retinoblastoma dephosphorylation and inhibited serum-induced AP-1 activation in young HDF, thus recapitulating basic biochemical and molecular changes of senescence. Sphingomyelinase and ceramide may thus be implicated as mediators of cellular senescence.     

Regulatory mechanisms of replication growth limits in cellular senescence

Normal human diploid fibroblasts cannot divide indefinitely in culture. At the end of their lifespan they withdraw from the cell cycle permanently by a process termed cellular senescence. Recent molecular studies indicate that upregulation of two inhibitors of cyclin-dependent kinases, p16 and p21, is responsible for blocking the G1/S transition in senescent cells. Although the state of senescence resembles terminal differentiation in that both exhibit irreversible growth arrest and resistance to apoptosis, other molecular changes are seen only in senescent cells. This suggests that the signal pathway specific for senescence is present in normal cells. Changes in chromosomes, such as progressive shortening of the telomeres and erosive damage by detrimental by-products in metabolism, may be the signals that trigger senescence, leading to the inactivation of cell cycle progression. On the other hand, it seems that a dominant genetic program is intrinsically preset to ensure a growth limit in the normal cell. This notion is supported by cell fusion and microcell transfer experiments which show that escaping from senescence requires recessive mutations in senescence-specific genes. Identification of these participating genes and clarification of their mode of action will provide the basis for understanding the mechanisms governing the differences between mortality in normal cells and immortality in cancer cells.     

Molecular and cellular basis of radiation fibrosis

From a spina bifida clinic we have identified two patients with a syndrome of myelomeningocele and Waardenburg syndrome type 3 (WS3). The patients each possess a single, de novo, interstitial deletion of chromosome 2 (2q35-36.2), including the PAX3 gene. Deletion of PAX3 was confirmed by fluorescence in situ hybridization (FISH). Analysis with PAX3 and flanking microsatellites shows that the deleted interval of chromosome 2 is of paternal origin and is at least 2 and 6 cM in the two patients. Interstitial deletions in this region result in the Waardenburg syndrome (WS1), but have not been associated with neural tube defects (NTDs). Although other etiologies have not been formally excluded, these patients raise the possibility of a digenic etiology of their NTDs via a genetic interaction of the deleted PAX3 gene with a second unidentified locus.     

Fibroblasts cultured from venous ulcers display cellular characteristics of senescence

PURPOSE: A well-recognized characteristic of venous ulcers is impaired healing. Fibroblasts cultured from venous ulcers (wound-fb) have been shown to have reduced growth rates and are larger than normal fibroblasts (normal-fb) from the ipsilateral limb. Reduced growth capacity and morphologic changes are 2 well-known traits of cellular senescence. Other molecular changes are overexpression of matrix proteins, such as cellular fibronectin (cFN), and enhanced activity of beta-galactosidase at pH of 6.0 (senescence associated beta-Gal, or SA-beta-Gal). Senescence, an irreversible arrest of cell proliferation with maintenance of metabolic functions, may represent in vivo aging and thus may be related to impaired healing. METHODS: Cultured normal-fb and wound-fb from 7 venous ulcer patients (average age, 51 years) were obtained by taking punch biopsies of the perimeter of the ulcer and from the ipsilateral thigh of the same patient. Growth rates, SA-beta-Gal activity, and level of cFN protein (immunoblot) and message (Northern blot) were measured. RESULTS: In all patients, wound-fb growth rates were significantly lower than those of normal-fb (P =.006). A higher percentage of SA-beta-Gal positive cells were found in all wound-fb (average, 6.3% vs. 0.21%; P =.016). The level of cFN, was consistently higher in all wound-fb tested. Also, in 4 patients, the level of cFN messenger RNA (mRNA) was increased. CONCLUSION: Fibroblasts cultured from venous ulcers exhibited characteristics associated with senescent cells. Accumulation of senescent cell in ulcer environment may be associated with impaired healing.     

Multiple pathways to cellular senescence: role of telomerase repressors

Telomeres progressively shorten with age in somatic cells in culture and in vivo because DNA replication results in the loss of sequences at the 5' ends of double-stranded DNA. Whereas somatic cells do not express the enzyme, telomerase, which adds repeated telomere sequences to chromosome ends, telomerase activity is detected in immortalised and tumour cells in vitro and in primary tumour tissues. This represents an important difference between normal cells and cancer cells, suggesting that telomere shortening causes cellular senescence. Hybrids between immortal cells and normal cells senesce, indicating that immortal cells have lost, mutated or inactivated genes that are required for the programme of senescence in normal cells. Genes involved in the senescence programme have been mapped to over ten different genetic loci using microcell fusion to introduce human chromosomes and restore the senescence programme. Multiple pathways of cellular senescence have also been demonstrated by chromosome transfer, indicating that the functions of the mapped senescence genes are probably different. One possibility is that one or more of these senescence genes may suppress telomerase activity in immortal cells, resulting in telomere shortening and cellular senescence. To test this hypothesis, telomerase activity and the length of terminal restriction fragments (TRFs) have been examined in microcell hybrids. Re-introduction of a normal chromosome 3 into the renal cell carcinoma cell line RCC23, which has the short arm of chromosome 3, restored cellular senescence. The loss of indefinite growth potential was associated with the loss of telomerase activity and shortening of telomeres in the RCC cells containing the introduced chromosome 3. However, microcell hybrids that escaped from senescence and microcell hybrids with an introduced chromosome 7 or 11 maintained telomere lengths and telomerase activity similar to the parental RCC23. Thus, restoration of cellular senescence by chromosome 3 is associated with repression of telomerase function in RCC cells. This evidence suggests that telomerase suppression is one of several pathways involved in immortalisation.     

Telomeres, not the end of the story

Transurethral needle ablation (TUNA) was performed on seven patients with chronic nonbacterial prostatitis who failed to respond to conventional treatments administered for more than half a year. The TUNA procedure heated the prostate to a temperature ranging from 90 degrees to 100 degrees C while the urethral temperature was maintained below 43 degrees C by a protective sheath and irrigation. After treatment, four patients showed complete resolution of symptoms and three a partial improvement. All patients had a decrease in the leukocyte count in expressed prostatic secretions (EPS) 1 month after treatment. Recurrence of abnormal inflammatory cells in the EPS was noted in two patients at 3 months after treatment. The subjective improvement has been maintained during the subsequent follow-up. From these results, TUNA is considered to be an effective, safe, and easy treatment for most patients with nonbacterial prostatitis.     

Clinical features associated with correction of T-cell senescence: increased acute-phase response, amyloidosis and arthritis

Two prominent features associated with immunosenescence are thymic involution and altered T-cell phenotype and responsiveness. We have shown previously that in CD2-fas transgenic mice, in which the Fas apoptosis molecule is constituatively expressed on T cells, T-cell senescence is greatly reduced. Using a different experimental approach, the relationship between T-cell senescence and apoptosis was analyzed on human PBMCs. The results indicate that there was increased apoptosis of CD45RO- (CD45RA+) T cells upon activation. We propose that this could account for the increase in CD45RO+ 'memory' T cells with aging in humans. Together these results are consistent with the notion that T-cell senescence is associated with altered apoptosis and decreased T-cell responsiveness. T-cell responsiveness remained high in CD2-fas transgenic aged mice, but there was no increase in overall life span of the mice. Increased T-cell responsiveness was associated with an increased acute-phase response and amyloid A deposition in the glomerulus of these mice. These data suggest that restoration of the T-cell immune function in aged individuals must be carried out in concert with correction of other immune factors that down modulate the acute-phase response to prevent undesirable side-effects.     

Early cell response to radiation

The early steps of cellular radiation response have been investigated using a linear electron accelerator operated in a split-dose mode, in such a way that the time intervals between pulse exposures to relativistic electrons ranged from fractions of a second to a few minutes. The initial dose brought about large, synchronous changes in radiation sensitivity and generated a tetraphasic, W-shaped time-dependent profile of cell survival upon the second radiation exposure. While this time-related process was observed in most cell lines investigated, its kinetic parameters varied significantly from one cell line to the other. The number of DNA strand breaks (neutral and alkaline DNA filter elution) and the level of apoptosis (gel electrophoresis and flow cytometry) induced at the different phases of the time-dependent profile showed no relationship with the W-effect. It is presently hypothesized that mechanisms involved in molecular recognition of radio-induced lesions and initiation of genomic instability play a major role in this effect. Whatever the mechanism involved, the split-dose irradiation in the range of seconds enables dissecting the early steps of radiation response. The relevance of the W-effect to radiation therapy and technical drawbacks are discussed.     

Demonstration of cellular aging and senescence in serially passaged long-term cultures of human trabecular osteoblasts

The proliferative capacity and cellular and biochemical characteristics of human trabecular bone osteoblasts were analysed throughout their replicative lifespan in vitro. Like several other cell types, human osteoblasts demonstrated a typical Hayflick phenomenon of cellular aging comprising a period of rapid proliferation until cumulative population doubling level (CPDL) 22 to 24, followed by a phase of slow growth and the final cessation of cell division at CPDL 32 to 34. Comparing young cells (less than 20% lifespan completed) and old cells (more than 90% lifespan completed) revealed a progressive increase in population doubling (PD) time, a decrease in attachment frequency, a decrease in the number of S-phase positive cells, a decrease in the rates of DNA, RNA and protein synthesis, an increase in the protein content per cell and an increased proportion of senescence-specific beta-galactosidase positive cells. While osteoblastic production of collagen type I decreased progressively during aging, alkaline phosphatase activity dropped rapidly after the first few passages and then remained constant during the rest of the proliferative lifespan, Significant morphological changes from thin and spindle-shaped early passage young cells to large, flattened and irregularly shaped late passage old cells full of intracellular debris were observed. In comparison, osteoblasts established from an osteoporotic bone sample showed a maximum CPDL of less than 5, had a longer PD time and exhibited abnormal senescent morphology. Thus, we have demonstrated for the first time that human osteoblasts, like several other diploid cell types, have a limited proliferative capacity in vitro and undergo aging and senescence as measured by various cellular and biochemical markers. In addition, preliminary studies show that cells from osteoporotic bone have a severely reduced proliferative capacity. This model of bone cell aging facilitates study of the molecular mechanisms of osteoblast senescence as well as factors related to osteoblast dysfunction in patients with osteoporosis.     

Thyroid hormone export regulates cellular hormone content and response

Actions of thyroid hormones (THs) are determined by intracellular free hormone concentration. Here we report that enhanced TH extrusion via a saturable, cold-sensitive mechanism lowers intracellular TH and causes TH resistance in hepatoma cells. Since these cells overexpress multidrug resistance P-glycoproteins and TH extrusion and resistance are blunted by verapamil, P-glycoproteins may mediate this resistance. Verapamil-inhibitable TH efflux was also found in primary hepatocytes, cardiocytes, and fibroblasts. These findings demonstrate that TH extrusion can modulate TH availability and action in mammalian cells.     

Telomeres and telomerase in normal and malignant haematologic cells

Tumor relapse remains a major obstacle to the success of allogeneic bone marrow transplantation (BMT) as a treatment for leukemia. Due to limited treatment options, the outlook for most patients that relapse following allogeneic BMT has been poor. The infusion of normal immunocompetent leukocytes from the original marrow donor has become a promising new option for treating/preventing leukemia relapse in allogeneic BMT recipients. This form of treatment has often been referred to as donor leukocyte infusion (DLI) therapy. Our laboratory is using murine models of allogeneic BMT to address important unresolved issues regarding DLI therapy in an effort to make the treatment more effective. These include identification of the antileukemic effector cells, augmentation of the antileukemic effect, and understanding why graft-versus-host-disease (GVHD) is less severe than anticipated. This article reviews our work in murine models of DLI and introduces our current working hypotheses concerning DLI therapy.     

SV40-transformed human cells in crisis exhibit changes that occur in normal cellular senescence

SV40 T antigen can induce senescent human diploid fibroblasts to synthesize DNA; however, the cells fail to go through mitosis. In this study, we examined the expression of the cdc2 and cyclin B genes, which are required for completion of mitosis, to determine whether defects in their expression occurred when SV40-transformed human cells entered the phase of crisis. If defects were observed it would indicate that immortalization by the virus involved reexpression of these genes. We found that the expression of cdc2 was unimpaired at both the RNA and protein levels, but that cyclin B expression was decreased in cells in crisis when compared with precrisis (mortal) and postcrisis (immortal) cells. Tritiated thymidine uptake demonstrated that the majority of cells in crisis were not actively cycling. Consistent with the latter observation we found that cyclin A, which is required for cells to traverse through S to G2, was downregulated in these cells. Since many of the results obtained with cells in crisis were similar to what is observed in normal human cells when they become senescent, we analyzed the expression of the genes fibronectin and sdi1 (a gene recently cloned from senescent cells that codes for an inhibitor of DNA synthesis). Both genes were overexpressed in cells during crisis, as is the case with senescent cells. The results are discussed in terms of the two-stage model previously proposed to explain the process of immortalization of human diploid fibroblasts by SV40.     

Aggresomes: a cellular response to misfolded proteins

Intracellular deposition of misfolded protein aggregates into ubiquitin-rich cytoplasmic inclusions is linked to the pathogenesis of many diseases. Why these aggregates form despite the existence of cellular machinery to recognize and degrade misfolded protein and how they are delivered to cytoplasmic inclusions are not known. We have investigated the intracellular fate of cystic fibrosis transmembrane conductance regulator (CFTR), an inefficiently folded integral membrane protein which is degraded by the cytoplasmic ubiquitin-proteasome pathway. Overexpression or inhibition of proteasome activity in transfected human embryonic kidney or Chinese hamster ovary cells led to the accumulation of stable, high molecular weight, detergent-insoluble, multiubiquitinated forms of CFTR. Using immunofluorescence and transmission electron microscopy with immunogold labeling, we demonstrate that undegraded CFTR molecules accumulate at a distinct pericentriolar structure which we have termed the aggresome. Aggresome formation is accompanied by redistribution of the intermediate filament protein vimentin to form a cage surrounding a pericentriolar core of aggregated, ubiquitinated protein. Disruption of microtubules blocks the formation of aggresomes. Similarly, inhibition of proteasome function also prevented the degradation of unassembled presenilin-1 molecules leading to their aggregation and deposition in aggresomes. These data lead us to propose that aggresome formation is a general response of cells which occurs when the capacity of the proteasome is exceeded by the production of aggregation-prone misfolded proteins.     

Distinction between differentiation and senescence and the absence of increased apoptosis in human keratinocytes undergoing cellular aging in vitro

OBJECTIVE: Changes in calcium homeostasis and bone mass around the climacteric are poorly understood. We examined relations between endocrine factors and indices of bone mass and metabolism in healthy women approaching the menopause. DESIGN: Cross-section study. PATIENTS: Sixty-eight spontaneously menstruating women aged 45-55. MEASUREMENTS: Bone density measured at lumbar spine (LS) and femoral neck (FN) using dual energy X-ray absorptiometry and distal non-dominant forearm using peripheral quantitative computed tomography. We recorded menstrual history, physical activity and dietary calcium, and measured serum calcium, phosphate, alkaline phosphatase, osteocalcin, vitamin D, fT3, T4, TSH, PTH, FSH and oestradiol (E2), and urinary pyridinoline (PYD) and deoxypyridinoline (DPD) excretion. RESULTS: Using serum FSH level as a marker of ovarian function, 63 subjects could be classified into one of three groups: group A (serum FSH < 10 U/l, n = 29), group B (10-35 U/l, n = 27) and group C (> 35 U/l, n = 7). Bone density fell with declining ovarian function at the LS, FN and forearm trabecular (but not cortical) sites. Serum PTH was lower in group A vs B (mean (SD) 2.68 (0.97) vs 3.52 (1.17) pmol/l, P < 0.05), but similar to group C (2.90 (1.09) pmol/l, P = NS). Serum phosphate was elevated in group C compared to groups A and B (1.17 (0.15) vs 1.04 (0.11) and 1.05 (0.13) mmol/l, P < 0.05), and urinary PYD (61.1 (8.0) vs 50.4 (11.6) and 43.9 (8.1) mumol/mol creatinine) and DPD (15.9 (3.9) vs 12.0 (3.6) and 11.4 (3.6) mumol/mol creatinine) excretion were also increased. There were no significant differences in vitamin D metabolites or osteocalcin. Multivariate analysis suggested serum osteocalcin was positively related to physical activity and serum 1,25-dihydroxycholecalciferol levels. Serum free T3 was positively correlated with urinary DPD excretion, and inversely related to serum PTH. In all subjects, serum PTH was related to body weight (r = 0.38, P = 0.002). CONCLUSIONS: Declining ovarian function before menopause is accompanied by reductions in bone mass and altered calcium metabolism. Free T3 may regulate bone resorption and indirectly modulate PTH release.