Critically short telomeres in acute myeloid leukemia with loss or gain of parts of chromosomes.

Telomeres, nucleoprotein complexes at chromosome ends, protect chromosomes against end-to-end fusion. Previous in vitro studies in human fibroblast models indicated that telomere dysfunction results in chromosome instability. Loss of telomere function can result either from critical shortening of telomeric DNA or from loss of distinct telomere-capping proteins. It is less clear whether telomere dysfunction has an important role in human cancer development in vivo. Acute myeloid leukemia (AML) is a good model to study mechanisms that generate chromosome instability in human cancer development because distinct groups of AML are characterized either by aberrations that theoretically could result from telomere dysfunction (terminal deletions, gains/losses of chromosome parts, nonreciprocal translocations), or aberrations that are unlikely to result from telomere dysfunction (e.g., reciprocal translocations or inversions). Here we demonstrate that AML with multiple chromosome aberrations that theoretically could result from telomere dysfunction is invariably characterized by critically short telomeres. Short telomeres in this group are not associated with low telomerase activity or decreased expression of essential telomeric capping proteins TRF2 and POT1. In contrast, telomerase activity levels are significantly higher in AML with short telomeres. Notably, short telomeres in the presence of high telomerase may relate to significantly higher expression of TRF1, a negative regulator of telomere length. Our observations suggest that, consistent with previous in vitro fibroblast models, age-related critical telomere shortening may have a role in generating chromosome instability in human AML development.

Telomerase activity level, but not hTERT mRNA and hTR level, regulates telomere length in telomerase-reconstituted primary fibroblasts.

The critical factors in the regulation of telomere length are not yet clearly defined. Telomerase is a key player in telomere elongation, although previous studies have shown that telomeres are differentially elongated after telomerase reconstitution. Moreover, a clear relation between the level of telomerase activity and telomere length was not observed. To investigate which factors are critical in telomere length regulation, we generated 24 telomerase-reconstituted primary human fibroblast clones. In these clones, in vitro telomerase activity level is clearly related to telomere length. High levels of telomerase activity are associated with longer telomeres and better telomere maintenance over time. The correlation coefficient, however, indicates that the level of telomerase activity is not the only factor in the regulation of telomere length. Clearly, factors that are not measured in an in vitro telomerase activity assay are involved in telomere length regulation in vivo. To investigate which telomerase components are critical in regulating telomerase activity levels, we studied expression levels of hTERT mRNA and hTR. Expression is highly variable between individual clones, but not related to the level of telomerase activity or telomere length. Our results indicate that expression levels of hTERT mRNA and hTR do not regulate the activity level of the telomerase complex, suggesting posttranscriptional modification of hTERT or the presence of additional proteins that modulate telomerase enzyme activity.

Granzyme expression in fine-needle aspirates from liver allografts is increased during acute rejection.

We investigated whether determination in fine-needle aspiration biopsy (FNAB) specimens of cells expressing granzymes (Grs) and Fas ligand would provide a reliable, easy, and quantitative measure of rejection activity in the transplanted liver. Retrospectively, 13 FNAB specimens obtained during clinical acute rejection, 10 FNAB specimens obtained during subclinical rejection, 12 FNAB specimens obtained during cytomegalovirus (CMV) infection, and 26 FNAB specimens obtained in the absence of rejection or infection were included on the study. Cytospin preparations of FNAB and peripheral-blood specimens were immunocytochemically stained for Fas-ligand and Gr, and increments in the liver were calculated by subtracting frequencies of positive cells in blood from those in FNAB specimens. Only sporadically Fas ligand-expressing, but many Gr-expressing, cells were detected in FNAB specimens. Increments in Gr-positive (Gr(+)) cells were significantly greater in FNAB specimens obtained during clinical rejection (median, 70 Gr(+) cells; range, 0 to 312 Gr(+) cells; P = .006) and tended to be greater in FNAB specimens obtained during subclinical rejection (median, 62 Gr(+) cells; range, 5 to 113 Gr(+) cells; P = .09) compared with those obtained in the absence of rejection (median, 16 Gr(+) cells; range, 0 to 103 Gr(+) cells). Increments obtained during clinical or subclinical rejection did not differ from those obtained during CMV infection (median, 27 Gr(+) cells; range, 6 to 212 Gr(+) cells). With the exclusion of specimens obtained during CMV infection, the sensitivity of Gr determination in FNAB specimens for the diagnosis of acute rejection (either clinical or subclinical) was 70%, and specificity, 69%. In FNAB specimens obtained during clinical and subclinical acute rejection episodes after liver transplantation, increased numbers of Gr-expressing cells were present; in the absence of CMV infection, their quantification provides a measure for rejection activity with moderate accuracy.