Telomeres, telomerase and cancer: is the magic bullet real?

Nature recruited telomerase to compensate for the incomplete replication of chromosomal ends (telomeres). In higher organisms, telomeres are eroded at each cell division. Cancer cells frequently show chromosomal instability resulting in ring chromosomes, telomeric associations, and dicentric chromosomes. As a consequence of telomeric erosion, the ribonucleoprotein complex termed "telomerase" is reactive in a subpopulation of cells. Telomerase adds a hexameric repeat of the sequence 5' TTAGGG 3' to the ends of the chromosomes and hence stabilizes the telomeric length. Telomerase is active in vertebrates mostly in germ cells and the early stage embryo but is inactivated or repressed in somatic cells. Detection of telomerase activity in the overwhelming majority of advanced and metastatic human cancers but not in most somatic cells implies that telomerase-dependent immortalization could contribute to the malignancy. Future studies on the expression and regulation of the individual components of telomerase may enable us to clarify the diagnostic and therapeutic potential of telomerase in cancer.

Prevalence and turnover of peptidylglycine alpha-amidating monooxygenase mRNA in atrial cardiomyocytes.

Peptidylglycine alpha-amidating monooxygenase (PAM), the enzyme responsible for the alpha-amidation of neuroendocrine peptides, is more prevalent in the atrium of the heart than in pituitary or brain. RNase protection assays indicate that PAM transcripts account for approximately 0.5% of the mRNA in the neonatal atrium and 0.06% of the mRNA in the neonatal ventricle. In primary atrial cardiomyocyte cultures PAM mRNA turns over slowly, with a half-life of approximately 20 h. Levels of PAM mRNA in primary atrial cardiomyocytes are increased to 16.5% of control upon treatment with dexamethasone and decreased to 63% of control upon treatment with thyroid hormone.

Antisense rel A in Cancer.

NFKB: is a pleiotropic transcription factor that participates in the induction of various cellular and viral genes (for a review, see refs. 1 and 2). The principal form of this active complex is composed of two polypeptides, p65 (recently renamed rel A) and p50 (recently renamed NFKB1) (3, 4). Both of these sub-units belong to the rel famtly of transcription factors with homology in the amino terminus (5, 6). Since the original identification of these factors, several others have been identified in this family of transcription factors: P49, c-rel, p100, rel B, dorsal, Bcl-3, and p105 (1). The NF-kheterodimer is associated in the cytoplasm with the IkB subunit, which sequesters this factor in an inactive state (7). On activation by various stimuli, the IkB subunit separates from the active NFheterodimer, and the active complex is translocated to the nucleus where it binds to its nuclear DNA target sequence (Fig. 1). The principal cause for disassociation of IkB from NFis phosphorylation (8), but it may involve proteolysis of IkB (9) Several autoregulatory loops have been proposed for the involvement of NF-kB in the regulation of the inhibitor IkB. Furthermore, NF-kB has been shown to regulate the transcription of IkB and NFKB1 (10-12). Fig. 1. NF-K: B transcription factor. This figure depicts the basis for the function of the NF-K: B transcription factor. The principal form of the NF-K: B transcription complex is as a heterodimer composed of the two subunits: rel A and NF-K: B 1. The inactive complex is sequestered in the cytoplasm associated with the inhibitor protein, IK: B. On activation, the IK: B protein separates from the active complex through a mechanism involving the phosphorylation and proteolysis Of IK: B. The active NF-K: B complex is then translocated to the nucleus where it binds DNA in a sequence-specific manner and interacts with the transcriptional machinery to modulate the rate of transcription of target genes.

Differentiation of immortal cells inhibits telomerase activity.

Telomerase, a ribonucleic acid-protein complex, adds hexameric repeats of 5'-TTAGGG-3' to the ends of mammalian chromosomal DNA (telomeres) to compensate for the progressive loss that occurs with successive rounds of DNA replication. Although somatic cells do not express telomerase, germ cells and immortalized cells, including neoplastic cells, express this activity. To determine whether the phenotypic differentiation of immortalized cells is linked to the regulation of telomerase activity, terminal differentiation was induced in leukemic cell lines by diverse agents. A pronounced downregulation of telomerase activity was produced as a consequence of the differentiated status. The differentiation-inducing agents did not directly inhibit telomerase activity, suggesting that the inhibition of telomerase activity is in response to induction of differentiation. The loss of telomerase activity was not due to the production of an inhibitor, since extracts from differentiated cells did not cause inhibition of telomerase activity. By using additional cell lineages including epithelial and embryonal stem cells, down-regulation of telomerase activity was found to be a general response to the induction of differentiation. These findings provide the first direct link between telomerase activity and terminal differentiation and may provide a model to study regulation of telomerase activity.

Sequence context of antisense RelA/NF-kappa B phosphorothioates determines specificity.

The use of antisense oligomers to achieve inhibition of gene expression is complicated by frequent non-specific effects, and even the control oligomers often exhibit sequence-specific effects. We have recently shown that in diverse tumor-derived cell lines, a 24mer phosphorothioate oligomer antisense to the relA subunit of NF-kappa B transcription factor causes a block of cellular adhesion, inhibition of nuclear NF-kappa B and Sp1 DNA-binding activity and inhibition of tumor cell growth in vitro and in vivo. In this study we use the same model to attempt to define the limits of antisense specificity. We demonstrate that single base pair substitution can virtually abolish the antisense activity. The relative position of mismatches within the antisense sequence is critical to the loss of activity. Our results further indicate that antisense specificity is determined not only by the content of the sequence but also by its occurrence with reference to the surrounding sequences.

Regulation of adhesion and growth of fibrosarcoma cells by NF-kappa B RelA involves transforming growth factor beta.

The NF-kappa B transcription factor is a pleiotropic activator that participates in the induction of a wide variety of cellular genes. Antisense oligomer inhibition of the RelA subunit of NF-kappa B results in a block of cellular adhesion and inhibition of tumor cell growth. Investigation of the molecular basis for these effects showed that in vitro inhibition of the growth of transformed fibroblasts by relA antisense oligonucleotides can be reversed by the parental-cell-conditioned medium. Cytokine profile analysis of these cells treated with relA antisense oligonucleotides revealed inhibition of transforming growth factor beta 1 (TGF-beta 1 to the transformed fibroblasts reversed the inhibitory effects of relA antisense oligomers on soft agar colony formation and cell adhesion to the substratum. Direct inhibition of TGF-beta 1 expression by antisense phosphorothioates to TGF-beta 1 mimicked the in vitro effects of blocking cell adhesion that are elicited by antisense relA oligomers. These results may explain the in vitro effects of relA antisense oligomers on fibrosarcoma cell growth and adhesion.

Maturation, internalization, and turnover of soluble and membrane proteins associated with atrial myocyte secretory granules.

Primary cultures of neonatal atrial myocytes were used to study the biosynthesis of a prominent secretory granule enzyme that occurs naturally in soluble and integral membrane forms. The two most prominent forms of peptidylglycine alpha-amidating monooxygenase PAM) in atrial myocytes are type I integral membrane proteins (PAM-1 and -2); smaller amounts of a soluble form, PAM-3, are also found. All three PAM proteins are N-glycosylated, and PAM-1 also has sialylated O-linked oligosaccharide. Two hours after their biosynthesis, approximately half of the newly synthesized PAM-1 and PAM-2 proteins have acquired N-linked oligosaccharide chains resistant to digestion with endoglycosidase-H. Secretion of newly synthesized PAM-3 is detectable within 90 min after biosynthesis and is largely complete within 4 h. Release of the catalytic domains of PAM-1 and PAM-2, which requires endoproteolytic cleavage, occurs at a slow rate for many hours after biosynthesis. Release of PAM-3 and the soluble PAM proteins derived from PAM-1 and PAM-2 can be stimulated by secretagogue. Integral membrane PAM proteins that reach the surface of atrial myocytes are internalized and enter the endocytic pathway. The turnover of newly synthesized PAM-1 and PAM-2 is only partially accounted for by the release of soluble PAM protein into the medium and may involve a significant contribution from intracellular degradation.

Developmental expression of peptidylglycine alpha-amidating monooxygenase (PAM) in primary cultures of neonatal rat cardiocytes: a model for studying regulation of PAM expression in the rat heart.

Primary cultures of neonatal rat atrial and ventricular cardiomyocytes were used to investigate the expression of peptidylglycine alpha-amidating monooxygenase (PAM), a bifunctional enzyme required for the production of alpha-amidated neuroendocrine peptides. The use of assays for the individual enzymes, peptidylglycine alpha-amidating monooxygenase (PHM) and peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL), demonstrated that the levels of expression observed in vitro approximated those observed in vivo. Both in vivo and in vitro, atrial and ventricular PAL activity greatly exceeded PHM activity. Atrial and ventricular cardiomyocytes secreted PHM and PAL activity at a constant rate throughout the culture period. Immunofluorescence studies localized PAM proteins to the perinuclear region, with intense punctate staining. Both in vivo and in vitro, PAM mRNAs encoding integral membrane proteins predominated throughout the neonatal period, with PAM-1 mRNA becoming more prevalent after the first week in culture. Although PAM-2 mRNA decreased in prevalence in vivo at the time when PAM-1 expression increased, levels of PAM-2 mRNA remained elevated throughout 2 weeks in vitro. Western blot analysis demonstrated intact PAM-1 and PAM-2 proteins in atrial cultures, with the prevalence of PAM-1 increasing in older cultures. Atrial cardiomyocytes secreted only bifunctional PAM proteins. Many of the features of PAM expression, processing, and storage that are unique to cardiomyocytes as opposed to endocrine cells are faithfully replicated by primary atrial and ventricular cultures.

Ontogeny of prolyl endopeptidase, pyroglutamyl peptidase I, TRH, and its metabolites in rat pancreas.

We demonstrate that two enzymes, soluble unspecific pyroglutamyl peptidase I and prolyl endopeptidase, able to degrade thyrotropin-releasing hormone (TRH) in vitro were present in pancreas at the early stage of rat development. Specific particulate pyroglutamyl peptidase II remained undetectable during ontogenesis. Pyroglutamyl peptidase I specific activity increased until day 3 and decreased after day 5. Furthermore, prolyl endopeptidase specific activity rose slightly to a peak on postnatal day 20. A good correlation between immunoreactive TRH and deaminated TRH (TRH-OH) was found in the 1st wk after birth. However, His-Pro diketopiperazine (DKP) levels were stable and low during development. We show that hot acidic extraction conditions could artefactually generate His-Pro DKP. In vivo, active site-directed inhibitors of pyroglutamyl peptidase I and prolyl endopeptidase enzymes do not show any TRH-deamidating and/or pyroglutamyl peptidase I pathways in neonatal rat pancreas. The data suggest that these two enzymes are not involved in intra- or extracellular control of TRH levels in neonatal rat pancreas and that pancreatic TRH content appears to be principally regulated by biosynthetic steps. Nevertheless, low levels of endogenous His-Pro DKP and TRH-OH identified in neonatal rat pancreas suggest that TRH or TRH-like peptides may be metabolized in this tissue in intact rats, albeit at low rates.

Peptidylglycine alpha-amidating monooxygenase activity and TRH and CRF biosynthesis. Role of copper.

Carboxy-terminal amidation of biologically active peptides, an important characteristic of more than half of these substances, occurs during the maturation process of peptide precursors. It is catalyzed by peptidylglycine alpha-amidating monooxygenase (PAM), an enzyme that is copper-dependent. We show here that alterations of copper stores in cultured cells from different origins (pancreas and hypothalamus) affect the immunoreactivity of thyrotropin-releasing hormone (TRH) and corticotropin-releasing factor (CRF) (two alpha-amidated peptides). This suggests that copper can affect neuropeptide biosynthesis and may play a role in the endocrine or central nervous system function.

Evidence for pyroglutamyl peptidase I and prolyl endopeptidase activities in the rat insulinoma cell line RINm 5F: lack of relationship with TRH metabolism.

Thyrotropin-Releasing hormone (TRH)-degrading pyroglutamyl peptidase I (PGP I) and prolylendopeptidase (PE) activities have been demonstrated in rat insulinoma RINm 5F cell line. These two enzymes catalyze the conversion of TRH to Histydyl-Proline-Diketopiperazine and to acid TRH respectively. After cell fractionation, we found all the PGP I and PE activities in the cytosolic fraction. The membrane-bound PGP II activity is not detectable in the RINm 5F cells. Further investigations on these two cytosolic enzymes show that pyroglutamyl- and proline-containing peptides are inhibitors of each TRH-degrading enzyme. Gel filtration chromatography on Sephadex G100 shows that PGP I and PE activity have an apparent molecular mass of about 18 kDa and 57 kDa, respectively. Kinetic analysis with TRH as substrate, gives a Km of 44 microM and 235 microM, and a Vmax of 1.49 and 8.80 pmol/min/micrograms protein for PGP I and PE, respectively. Immunoreactive TRH, His-Pro-Diketopiperazine and acid TRH levels in the cell line extracts are 2.2 +/- 0.9, 22.5 +/- 11.1 and 28.7 +/- 14.6 pg/1O6 cells, respectively. When cells have been incubated for 2 to 72 hours with a P.E. inhibitor (Z-Gly-Pro-CHN2) at 5 x 10(-7) M, both cell PGP I and PE activities are inhibited. No change in the cellular content of immunoreactive TRH, His-Pro-Diketopiperazine and acid TRH have been observed in treated cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Glucocorticoids regulate peptidyl-glycine alpha-amidating monooxygenase gene expression in the rat hypothalamic paraventricular nucleus.

Peptidyl-glycine alpha-amidating monooxygenase (PAM) is a posttranslational processing enzyme which catalyzes the formation of biologically active alpha-amidated peptides. The two major neuropeptides involved in the regulation of ACTH secretion [CRF and arginine vasopressin (AVP)], synthesized in the parvocellular part of the hypothalamic paraventricular nucleus (PVN), are amidated, and their synthesis and/or release is negatively regulated by glucocorticoids. In this study, using in situ hybridization, we have shown that PAM mRNA is abundantly expressed in the hypothalamic paraventricular and supraoptic nucleus. Surgical adrenalectomy (ADX) induced increases in PAM, CRF, and AVP mRNA in the parvocellular part of the PVN, while corticosterone treatment normalized these values. PAM and AVP gene expression were not changed in the magnocellular part of the PVN or in the supraoptic nucleus. These observations suggest that in addition to stimulation of CRF and AVP synthesis, ADX induces an increase in PAM synthesis in the PVN and, thus, support the hypothesis of increased secretion of both CRF and AVP after ADX.

Regulation of TRH release by the cultured neonate rat pancreas.

The TRH secretory responsiveness of the pancreatic islet cell clusters from newborn rat in organ culture was studied. Basal TRH secretion was stable over a 9-day period. The response to various secretagogues was tested on day 4. TRH secretion was stimulated by high potassium-induced depolarization and also through both cAMP and protein kinase-C dependent pathways. Like insulin, TRH release was stimulated by glucose and arginine and inhibited by somatostatin. These data suggest the existence of a common mechanism for TRH and insulin secretion by the pancreatic beta-cells.

Characterization of an alpha-amidating activity in a human pancreatic tumour secreting vasoactive intestinal peptide (VIP).

A case of watery diarrhoea hypokalaemia achlorhydria (WDHA) syndrome due to a pancreatic tumour and identified by VIP plasma level, VIP immunocytochemistry, and ultrastructural analysis of tumour sections, is reported. Since VIP is the mediator of the syndrome and is biologically active under its amidated form, the enzymatic alpha-amidating activity was investigated and characterized in tumour extract; using the synthetic substrate D-Tyr-Val-Gly, the enzyme displayed an optimal activity at pH 7.0, under aerobic conditions and with 35 microM CuSO4 and 3 mM ascorbate as co-factors. The Kmax and Vmax values of the enzymatic activity were 133.7 microM and 26.9 pmol/h/micrograms protein respectively. Its molecular weight, determined by molecular sieving, was close to 36 kDa. Other tumours of the human endocrine pancreas were also investigated for the enzymatic activity. The clinical interest of studying the regulation of the alpha-amidating activity in such tumours is discussed.

Ontogenetic expression of peptidyl-glycine alpha-amidating monooxygenase mRNA in the rat pancreas.

Qualitative and quantitative expression of m.RNA coding for Peptidyl-Glycine alpha-Amidating Monooxygenase (PAM) in the developing rat pancreas was investigated by Northern and dot blot hybridization, with a bovine PAM c.DNA probe (0.7 kb fragment). A specific hybridization signal was evidenced for a 3.7 kb m.RNA species. Measurement of PAM m. RNA rate during the rat pancreas ontogenesis revealed a biphasic profile which appeared corelated with that of gastrin and TRH m.RNA respectively. On the other hand, streptozotocin-treatment resulted in a 50% decrease of PAM m.RNA levels.