The role of the cell attachment in the regulation of telomerase during keratinocyte differentiation

PhDThe catalytic subunit of telomerase TERT has non-canonical functions, which are independent of telomere elongation and sometimes telomerase activity. In skin keratinocytes, both TERT expression and anoikis are regulated by extracellular matrix molecules and their integrin receptors, but the effect of TERT deregulation on anoikis has not been previously investigated. HaCaT cells expressing wild-type TERT, TERT-HA (non-canonical function only) DnTERT (catalytically inactive TERT) and the empty vector (PURO) were created by retroviral transduction. HaCaT anoikis was monitored after disengagement of integrins following anchorage deprivation by nuclear staining and FACS. The expression of both TERT and TERT-HA, and to a lesser extent DnTERT, significantly decreased the appearance of apoptotic HaCaT cells in suspension. This showed that TERT could mute anoikis even in the absence of telomere lengthening but that it may require telomerase activity for optimum effect. TERT/TERT-HA did not mute cisplatin-induced HaCaT apoptosis, suggesting that the effects are specific to anoikis. Telomerase activity in the anchorage-deprived samples significantly decreased compared to the controls at time zero in all the samples. hTERT mRNA expression dropped in all groups in suspension compared to the zero time empty vector controls but the expression was still higher in those cells over expressing TERT/TERT-HA than the controls at zero time, suggesting a relationship between TERT/TERT-HA expression and resistance to anoikis. However, TERC expression increased in suspension similarly in all experimental groups. Therefore, these results suggest that the downregulation of TERT and not TERC contributes to the downregulation of telomerase in suspension, although the downregulation of ectopically expressed TERT also suggests that post-transcriptional mechanisms are involved. TERT splice variants were rare and not strikingly regulated in suspended keratinocytes. Western blot and FACS analyses indicated that TERT does not act by affecting integrin expression or their density on the keratinocyte surface, suggesting a point of action downstream of integrin

Crypt fusion as a homeostatic mechanism in the human colon.

OBJECTIVE: The crypt population in the human intestine is dynamic: crypts can divide to produce two new daughter crypts through a process termed crypt fission, but whether this is balanced by a second process to remove crypts, as recently shown in mouse models, is uncertain. We examined whether crypt fusion (the process of two neighbouring crypts fusing into a single daughter crypt) occurs in the human colon. DESIGN: We used somatic alterations in the gene cytochrome c oxidase (CCO) as lineage tracing markers to assess the clonality of bifurcating colon crypts (n=309 bifurcating crypts from 13 patients). Mathematical modelling was used to determine whether the existence of crypt fusion can explain the experimental data, and how the process of fusion influences the rate of crypt fission. RESULTS: In 55% (21/38) of bifurcating crypts in which clonality could be assessed, we observed perfect segregation of clonal lineages to the respective crypt arms. Mathematical modelling showed that this frequency of perfect segregation could not be explained by fission alone (p<10-20). With the rates of fission and fusion taken to be approximately equal, we then used the distribution of CCO-deficient patch size to estimate the rate of crypt fission, finding a value of around 0.011 divisions/crypt/year. CONCLUSIONS: We have provided the evidence that human colonic crypts undergo fusion, a potential homeostatic process to regulate total crypt number. The existence of crypt fusion in the human colon adds a new facet to our understanding of the highly dynamic and plastic phenotype of the colonic epithelium.wellcome trust royal societ

The mutational landscape of the adult healthy parous and nulliparous human breast

Abstract The accumulation of somatic mutations in healthy human tissues has been extensively characterized, but the mutational landscape of the healthy breast is still poorly understood. Our analysis of whole-genome sequencing shows that in line with other healthy organs, the healthy breast during the reproduction years accumulates mutations with age, with the rate of accumulation in the epithelium of 15.24 ± 5 mutations/year. Both epithelial and stromal compartments contain mutations in breast-specific driver genes, indicative of subsequent positive selection. Parity- and age-associated differences are evident in the mammary epithelium, partly explaining the observed difference in breast cancer risk amongst women of different childbearing age. Parity is associated with an age-dependent increase in the clone size of mutated epithelial cells, suggesting that older first-time mothers have a higher probability of accumulating oncogenic events in the epithelium compared to younger mothers or nulliparous women. In conclusion, we describe the reference genome of the healthy female human breast during reproductive years and provide evidence of how parity affects the genomic landscape of the mammary gland

The mutational landscape of the adult healthy parous and nulliparous human breast

The accumulation of somatic mutations in healthy human tissues has been extensively characterized, but the mutational landscape of the healthy breast is still poorly understood. Our analysis of whole-genome sequencing shows that in line with other healthy organs, the healthy breast during the reproduction years accumulates mutations with age, with the rate of accumulation in the epithelium of 15.24 ± 5 mutations/year. Both epithelial and stromal compartments contain mutations in breast-specific driver genes, indicative of subsequent positive selection. Parity- and age-associated differences are evident in the mammary epithelium, partly explaining the observed difference in breast cancer risk amongst women of different childbearing age. Parity is associated with an age-dependent increase in the clone size of mutated epithelial cells, suggesting that older first-time mothers have a higher probability of accumulating oncogenic events in the epithelium compared to younger mothers or nulliparous women. In conclusion, we describe the reference genome of the healthy female human breast during reproductive years and provide evidence of how parity affects the genomic landscape of the mammary gland.</p