Hector F. Valenzuela

Telomere shortening in T cells correlates with Alzheimer’s disease status

Telomeres, the repeated sequences that cap chromosome ends, undergo shortening with each cell division, and therefore serve as markers of a cells replicative history. In vivo, clonal expansion of T cells during immune responses to both foreign and autoantigens is associated with telomere shortening. To investigate possible immune alterations in Alzheimers disease (AD) that might impact current vaccine-based therapeutic strategies, we analyzed telomere lengths in immune cell populations from AD patients. Our data show a significant telomere shortening in PBMC from AD versus controls (P=0.04). Importantly, telomere length of T cells, but not of B cells or monocytes, correlated with AD disease status, measured by Mini Mental Status Exam (MMSE) scores (P=0.025). T cell telomere length also inversely correlated with serum levels of the proinflammatory cytokine TNFalpha (a clinical marker of disease status), with the proportion of CD8+ T cells lacking expression of the CD28 costimulatory molecule, and with apoptosis. These findings suggest an immune involvement in AD pathogenesis.

O-Glycosylation Regulates LNCaP Prostate Cancer Cell Susceptibility to Apoptosis Induced by Galectin-1

Resistance to apoptosis is a critical feature of neoplastic cells. Galectin-1 is an endogenous carbohydrate-binding protein that induces death of leukemia and lymphoma cells, breast cancer cells, and the LNCaP prostate cancer cell line, but not other prostate cancer cell lines. To understand the mechanism of galectin-1 sensitivity of LNCaP cells compared with other prostate cancer cells, we characterized glycan ligands that are important for conferring galectin-1 sensitivity in these cells, and analyzed expression of glycosyltransferase genes in galectin-1-sensitive, prostate-specific antigen-positive (PSA(+)) LNCaP cells compared with a galectin-1-resistant PSA(-) LNCaP subclone. We identified one glycosyltransferase, core 2 N-acetylglucosaminyltransferase, which is down-regulated in galectin-1-resistant PSA(-) LNCaP cells compared with galectin-1-sensitive PSA(+) LNCaP cells. Intriguingly, this is the same glycosyltransferase required for galectin-1 susceptibility of T lymphoma cells, indicating that similar O-glycan ligands on different polypeptide backbones may be common death trigger receptors recognized by galectin-1 on different types of cancer cells. Blocking O-glycan elongation by expressing alpha2,3-sialyltransferase 1 rendered LNCaP cells resistant to galectin-1, showing that specific O-glycans are critical for galectin-1 susceptibility. Loss of galectin-1 susceptibility and synthesis of endogenous galectin-1 has been proposed to promote tumor evasion of immune attack; we found that galectin-1-expressing prostate cancer cells killed bound T cells, whereas LNCaP cells that do not express galectin-1 did not kill T cells. Resistance to galectin-1-induced apoptosis may directly contribute to the survival of prostate cancer cells as well as promote immune evasion by the tumor.

In vitro senescence of immune cells

Immune cells are eminently suitable model systems in which to address the possible role of replicative senescence during in vivo aging. Since there are more than 10(8) unique antigen specificities present within the total T lymphocyte population of each individual, the immune response to any single antigen requires massive clonal expansion of the small proportion of T cells whose receptors recognize that antigen. The Hayflick Limit may, therefore, constitute a barrier to effective immune function, at least for those T cells that encounter their specific antigen more than once over the life course. Application of the fibroblast replicative senescence model to the so-called cytotoxic or CD8 T cell, the class of T cells that controls viral infection and cancer, has revealed certain features in common with other cell types as well as several characteristics that are unique to T cells. One senescence-associated change that is T cell-specific is the complete loss of expression of the activation signaling surface molecule, CD28, an alteration that enabled the documentation of high proportions of senescent T cells in vivo. The T cell model has also provided the unique opportunity to analyze telomere dynamics in a cell type that has the ability to upregulate telomerase yet nevertheless undergoes senescence. The intimate involvement of the immune system in the control of pathogens and cancer as well as in modulation of bone homeostasis suggests that more extensive analysis of the full range of characteristics of senescent T cells may help elucidate a broad spectrum of age-associated physiological changes.

Functional assessment of pharmacological telomerase activators in human T cells.

Telomeres are structures at the ends of chromosomes that shorten during cell division and eventually signal an irreversible state of growth arrest known as cellular senescence. To delay this cellular aging, human T cells, which are critical in the immune control over infections and cancer, activate the enzyme telomerase, which binds and extends the telomeres. Several different extracts from the Astragalus membranaceus root have been documented to activate telomerase activity in human T cells. The objective of this research was to compare two extracts from Astragalus membranaceus, TA-65 and HTA, for their effects on both telomerase and proliferative activity of human CD4 and CD8 T cells. Our results demonstrate that, TA-65 increased telomerase activity significantly (1.3 to 3.3-fold relative to controls) in T cell cultures from six donors tested, whereas HTA only increased telomerase levels in two out of six donors. We also demonstrate that TA-65 activates telomerase by a MAPK- specific pathway. Finally, we determine that during a three-day culture period, only the T cells treated with the TA-65 extract showed a statistically significant increase in proliferative activity. Our results underscore the importance of comparing multiple telomerase activators within the same experiment, and of including functional assays in addition to measuring telomerase activity.

Telomeres and Replicative Senescence

Telomere length measurement can be used both to monitor the proliferation of long-term cultures of somatic cells as well as to determine the replicative history of in vivo-derived cells. The most frequently used technique for telomere length measurement is Southern hybridization (1, 4). The method consists of isolating total genomic DNA, digesting the DNA with restriction enzymes so as to isolate the undigested telomere restriction fragments (TRFs), and separating these fragments by gel electrophoresis. The DNA is denatured and transferred from the gel to a membrane or filter, and the DNA samples are then hybridized to radiolabeled complementary probe. However, when blotting TRF DNA to the membrane, differential transfer may occur owing to inefficient transfer of larger fragments of DNA (>10 kb) to a membrane. As the mean length of the TRF is based on the assumption that the amount of telomeric DNA (TTAGGG repeats) in a given TRF is proportional to the length (3, 4), this would lead to possible error in calculating the mean length of the telomeres. The method that we present here avoids these potential problems by eliminating the membrane blot step altogether and probing the gel directly.

The Aging Lymphocyte

The life expectancy in industrialized countries has consistently increased every decade for the last 150 years. From the early 1900s to 2006, the United States alone experienced an increase in life expectancy from 49.2 to 77.7 years [1]. Although most of these increases have occurred in industrialized countries, the world as a whole has also seen improvements. It is projected that, by the year 2025, there will be more than one billion people over the age of 60 worldwide (see World Health Organization website). Increases in life expectancy reflect advances in both public health policy and improved treatments for infectious diseases. However, the increases in average human lifespan also present growing challenges to a nation’s economic, medical, social fabric, and public health programs. Older age is associated with a significantly higher incidence of various chronic conditions, such as cardiovascular disease, cancer, diabetes, neuropathologies, and immune system dysfunction. Persons with these conditions constitute a large burden on the health care system, and in many cases, the lack of adequate treatments have negative effects on quality of life and independent living. Thus, improved understanding of the fundamental molecular basis of aging is critical in order to adequately address the health of an increasingly large proportion of the population.