Naotoshi Kanda

Telomerase overexpression in K562 leukemia cells protects against apoptosis by serum deprivation and double-stranded DNA break inducing agents, but not against DNA synthesis inhibitors

Telomeres are specialized DNA/protein structures that act as protective caps to prevent end fusions. The maintenance of telomeres is essential for chromosomal stability. Telomerase is regulated by human telomerase reverse transcriptase (hTERT). c-Myc oncoprotein is also implicated in the positive regulation of hTERT expression. We show here that two clones of hTERT-transfected K562 erythroleukemia cells have elongated telomeres (22.5 and 24.0 kb), whereas telomere length of both c-Myc-transfected K562 cells and parental K562 cells is 6.5 kb. Telomerase activity and hTERT mRNA expression increased in hTERT-transfected K562 cells, while the expression levels of telomerase activity and hTERT in c-Myc-transfected K562 cells were similar to that in parental K562 cells, despite an overexpression of c-Myc. Importantly, we found that hTERT-transfected K562 cells are protected against apoptosis induced by serum deprivation and double-stranded DNA break inducing agents (ionizing irradiation, and etoposide (VP-16)), but not against DNA synthesis inhibitors (1-beta-D-arabinofuranosylcytosine and hydroxyurea). These findings suggest that overexpression of telomerase by transfecting hTERT confers telomere-elongation and resistance to double-stranded DNA break inducing agents.

A major determinant quantitative-trait locus responsible for atopic dermatitis-like skin lesions in NC/Nga mice is located on Chromosome 9.

Abstract. NC/Nga (NC) is a newly discovered model mouse for human atopic dermatitis, NC mice showing specific symptoms such as dermatitis and overproduction of IgE. To detect the loci responsible for the onset of dermatitis in the mice, backcross (N2) progeny between (NC×MSM/MS)F1 and NC were generated, where MSM/MS is an inbred strain from Japanese wild mice, Mus musculus molossinus. Linkage disequilibrium between dermatitis and various chromosome-specific microsatellite markers was then examined in the N2 segregants with severe dermatitis. The analysis revealed that the locus of the major determinant (designated here as derm1) was tightly linked to D9Mit163, D9Mit72, D9Mit143, D9Mit103, D9Mit207, and D9Mit209, because these markers showed the highest and most significant χ2 values. Since no recombination was observed among the markers in our linkage map, a radiation hybrid (RH) panel was applied to locate the derm1 locus more precisely. The markers were separated on the RH map, and their order was D9Mit163–D9Mit72–D9Mit143–D9Mit103–D9Mit207–D9Mit209 from the centromere. Several functional candidate genes are located near the locus derm1. These candidates are Thy1, Cd3d, Cd3e, Cd3g, Il10ra, Il18, and Csk, all of which could be involved in allergic responses through effects on T-cell function. Of these candidates, Csk is the strongest for NC dermatitis, since its map position was most tightly linked to the derm1 locus.

Klotho insufficiency causes decrease of ribosomal RNA gene transcription activity, cytoplasmic RNA and rough ER in the spinal anterior horn cells

The klotho gene was identified in 1997 as the gene whose severe insufficiency (kl/kl) causes a syndrome resembling human aging, such as osteoporosis, arteriosclerosis, gonadal atrophy, emphysema, and short life span in a mouse strain. Regarding the gait disturbance reported in kl/kl mice, the present study examined the spinal cord of kl/kl mice, and revealed decreases in the number of large anterior horn cells (AHCs), the amount of cytoplasmic RNA, the number of ribosomes and rough endoplasmic reticulum (rER), and the activity of ribosomal (r) RNA gene transcription without significant loss of the total number of neurons in the ventral gray matter. Increased immunostaining of phosphorylated neurofilament in the AHCs and of glial fibrillary acidic protein in reactive astrocytes in the anterior horn of kl/kl mice were also observed. On the other hand, the posterior horn was quite well preserved. The results suggest that the kl/kl insufficiency causes atrophy and dysfunction of the spinal AHCs through decreased activity of rRNA gene transcription, which may reduce the amount of cytoplasmic RNA and the number of ribosomes and rER. These findings resemble those found in the spinal cord of patients with classic amyotrophic lateral sclerosis (ALS). The results show that klotho gene insufficiency causes dysfunction of the protein synthesizing system in the AHCs, and might indicate the klotho gene is involved in the pathological mechanism of classic ALS. The kl/kl is a new animal model of AHC degeneration, and may provide clues to understanding the etiology of classic ALS.

Overexpression of telomerase confers a survival advantage through suppression of TRF1 gene expression while maintaining differentiation characteristics in K562 cells.

Leukemic stem cells that expressed endogenous telomerase activity were induced to show overexpression of exogenous hTERT and were analyzed for biological changes in order to assess the possible influence of telomerase gene therapy on the transplantation of normal hematopoietic stem cells. Introduction of hTERT into K562, a telomerase-positive immortal cell line, resulted in a 2.5-fold elevation of telomerase activity and the lengthening of telomeres by 6 kb to 23 kb. Real-time fluorescent PCR, which could perform quantitative analysis of transcripts, revealed a 175-fold increase in hTERT expression, suggesting the posttranscriptional regulation of telomerase. Ectopic expression of hTERT in K562 cells showed a survival advantage during culture in the absence of serum. Expression of mRNA for the telomeric-repeat binding factor 1 (TRF1) and caspase-3 activity were both decreased in hTERT-transfected K562 cells. Transduced cells retained their usual phenotypic characteristics, differentiation ability, and signal transduction response to TPA. These data suggest that ectopic expression of hTERT by normal hematopoietic stem cells may confer a survival advantage without changing their innate biological characteristics.

c-myb gene analysis in T-cell malignancies with del(6q)

Three T-cell malignancies with del(6q) were analyzed for karyotypes and alteration of the oncogene c-myb that is assigned to 6q22-q24. Patients were diagnosed as having non-Hodgkin T-cell lymphoblastic lymphoma, adult T-cell leukemia, and acute T-cell lymphoblastic leukemia, and the deletions of chromosome 6 were del(6)(q21q25), del(6)(q21q23), and del(6)(q21) or del(6)(q21q27), respectively. Tumor cell DNAs were obtained from cultured pleural fluid or from fresh peripheral blood and marrow samples and were analyzed by Southern blot hybridization, using c-myb oncogene probes. Rearrangements, deletions, or amplifications were absent in these tumor DNAs, thereby indicating that the del(6q) breakpoint in these T-cell malignancies was located outside of the c-myb gene. Northern blot analysis revealed the elevated expression of c-myb in the non-Hodgkin lymphoma patient, in accord with lineage characteristics.

Donor age reflects the replicative lifespan of human fibroblasts in culture

Human fibroblasts, which have a finite lifespan in cultures, have been widely used as a model system for cellular aging, and frequently used as one model of human aging. But whether cellular aging contributes to organismal aging has been controversial. To reinvestigate this question, we cultured human fibroblasts from the skin of one individual volunteer collected at different ages. Over a period of 27 years (donor age 36 years to 62 years), we obtained skin cells four times at appropriate intervals, and established eight fibroblast lines. These human fibroblasts have presented evidence for a correlation between donor age and proliferative lifespan in vitro. This result parallels the fact that telomeric DNA size cultured fibroblasts decrease with the increase in donor age. These cell lines had a normal diploid human chromosome constitution and will be useful in studies of human biology including aging.