mTOR signaling: implications for cancer and anticancer therapy.

Mounting evidence links deregulated protein synthesis to tumorigenesis via the translation initiation factor complex eIF4F. Components of this complex are often overexpressed in a large number of cancers and promote malignant transformation in experimental systems. mTOR affects the activity of the eIF4F complex by phosphorylating repressors of the eIF4F complex, the eIF4E binding proteins. The immunosuppressant rapamycin specifically inhibits mTOR activity and retards cancer growth. Importantly, mutations in upstream negative regulators of mTOR cause hamartomas, haemangiomas, and cancers that are sensitive to rapamycin treatment. Such mutations lead to increased eIF4F formation and consequently to enhanced translation initiation and cell growth. Thus, inhibition of translation initiation through targeting the mTOR-signalling pathway is emerging as a promising therapeutic option.

mTOR, translation initiation and cancer.

Control of mRNA translation plays a fundamental role in many aspects of cell metabolism. It constitutes a critical step in the control of gene expression, and consequently cell growth, proliferation and differentiation. Translation is regulated in response to nutrient availability, hormones, mitogenic and growth factor stimulation and is coupled with cell cycle progression and cell growth. Signaling by the PI3K/Akt/mTOR pathway profoundly affects mRNA translation through phosphorylation of downstream targets such as 4E-BP and S6K. Inhibitors of this pathway and thus cap-dependent translation are emerging as promising therapeutic options for the treatment of cancer.

mTOR signaling: implications for cancer and anticancer therapy.

Mounting evidence links deregulated protein synthesis to tumorigenesis via the translation initiation factor complex eIF4F. Components of this complex are often overexpressed in a large number of cancers and promote malignant transformation in experimental systems. mTOR affects the activity of the eIF4F complex by phosphorylating repressors of the eIF4F complex, the eIF4E binding proteins. The immunosuppressant rapamycin specifically inhibits mTOR activity and retards cancer growth. Importantly, mutations in upstream negative regulators of mTOR cause hamartomas, haemangiomas, and cancers that are sensitive to rapamycin treatment. Such mutations lead to increased eIF4F formation and consequently to enhanced translation initiation and cell growth. Thus, inhibition of translation initiation through targeting the mTOR-signalling pathway is emerging as a promising therapeutic option.

Parathyroid hormone-related protein promotes quiescence and survival of serum-deprived chondrocytes by inhibiting rRNA synthesis.

Parathyroid hormone-related protein (PTHrP) was initially recognized for its ability to promote parathyroid hormone-like bioactivity in kidney, bone, and squamous epithelial cells. PTHrP is a multifunctional protein in which bioactivity is mediated by two distinct pathways. Its classic parathyroid hormone-like activity results from binding of its amino terminus to cell surface PTH1R and activation of signal transduction pathways. Another less well recognized pathway involves translocation of PTHrP to the nucleus via a mid-region bipartite nuclear targeting sequence (NTS), similar in structure and function to those found in retroviral regulatory proteins. PTHrP was identified in the nucleus of several different cell types in vivo and in vitro, where it has been implicated in cell cycle progression, cellular differentiation, and apoptosis. In previous work we showed that nuclear translocation of PTHrP enhanced the survival of serum-deprived chondrogenic cells, associated with RNA, and localized to a region of the nucleus rich in complexes of newly transcribed ribosomal RNA and protein. In this work we have used two chondrogenic cell lines, CFK2 (PTH1R+) and 27m21 (PTH1R-) to further explore mechanisms whereby PTHrP rescues immature chondrocytes from apoptosis. Endogenous PTHrP and exogenous PTHrP NTS peptide protected serum-deprived cells from apoptosis, in the presence and absence of PTH1R. The survival of cells expressing PTHrP and those treated with PTHrP NTS peptide was associated with a rapid shift into G(o)/G1 accompanied by a significant down-regulation of rRNA synthesis and a decrease in the number of actively translating polyribosome complexes. Together with our previous observations, this work predicts a role for PTHrP in modulating ribosome biogenesis and preventing chondrogenic cells from progressing through the cell cycle in an unfavorable environment.

Characterization of elongation factor-1A (eEF1A-1) and eEF1A-2/S1 protein expression in normal and wasted mice.

The eEF1Alpha-2 gene (S1) encodes a tissue-specific isoform of peptide elongation factor-1A (eEF1A-1); its mRNA is expressed only in brain, heart, and skeletal muscle, tissues dominated by terminally differentiated, long-lived cells. Homozygous mutant mice exhibit muscle wasting and neurodegeneration, resulting in death around postnatal day 28. eEF1Alpha-2/S1 protein shares 92% identity with eEF1A-1; because specific antibodies for each were not available previously, it was difficult to study the developmental expression patterns of these two peptide elongation factors 1A in wasted and wild-type mice. We generated a peptide-derived antiserum that recognizes the eEF1Alpha-2/S1 isoform and does not cross-react with eEF1A-1. We characterized the expression profiles of eEF1A-1 and eEF1A-2/S1 during development in wild-type (+/+), heterozygous (+/wst), and homozygous (wst/wst) mice. In wild-type and heterozygous animals, eEF1A-2/S1 protein is present only in brain, heart, and muscle; the onset of its expression coincides with a concomitant decrease in the eEF1A-1 protein level. In wasted mutant tissues, even though eEF1A-2/S1 protein is absent, the scheduled decline of eEF1A-1 occurs nonetheless during postnatal development, as it does in wild-type counterparts. In the brain of adult wild-type mice, the eEF1A-2/S1 isoform is localized in neurons, whereas eEF1A-1 is found in non-neuronal cells. In neurons prior to postnatal day 7, eEF1A-1 is the major isoform, but it is later replaced by eEF1A-2/S1, which by postnatal day 14 is the only isoform present. The postdevelopmental appearance of eEF1A-2/S1 protein and the decline in eEF1A-1 expression in brain, heart, and muscle suggest that eEF1A-2/S1 is the adult form of peptide elongation factor, whereas its sister is the embryonic isoform, in these tissues. The absence of eEF1A-2/S1, as well as the on-schedule development-dependent disappearance of its sister gene, eEF1A, in wst/wst mice may result in loss of protein synthesis ability, which may account for the numerous defects and ultimate fatality seen in these mice.

Signaling pathway for apoptosis: a racetrack for life or death.

Apoptosis, or programmed cell death, is a gene-directed mechanism activated as a suicidal event to get rid of excess, damaged, or infected cells. The recent astounding pace of research in this area has expanded our horizon of understanding that this mechanism is regulated largely by pro- and anti-apoptosis factors acting for or against the final death event. The driving force behind these factors, either pro-apoptosis or pro-survival, is largely determined by signal transduction pathways, starting with the initiation of a death signal at the plasma membrane, and following through a complex cytoplasmic network before reaching the end point of cell demise. Enmeshed in this intricate cytoplasmic network are many checkpoints, where complexes of pro- and anti-apoptosis factors function to facilitate or deter the death signals. The culmination of the balancing act between these two camps of factors at these signal transduction checkpoints may then result in the final decision to die or to live. Thus, the eventual death of a cell may require successful passage through all the checkpoints, a mechanism Nature has provided as a safeguard to prevent erroneous triggering of death. With the advent of a new biotechnology revolution at the dawn of the new millenium, we look forward to an exciting era when we can gain fuller understanding of the operation of all these checkpoints. Ultimately, this gain will pave the way to control the apoptosis event at the checkpoints, and to support the organism's functionality as long as possible. J. Cell. Biochem. Suppls. 32/33:95-102, 1999.

W474C amino acid substitution affects early processing of the alpha-subunit of beta-hexosaminidase A and is associated with subacute G(M2) gangliosidosis.

Mutations in the HEXA gene, encoding the alpha-subunit of beta-hexosaminidase A (Hex A), that abolish Hex A enzyme activity cause Tay-Sachs disease (TSD), the fatal infantile form of G(M2) gangliosidosis, Type 1. Less severe, subacute (juvenile-onset) and chronic (adult-onset) variants are characterized by a broad spectrum of clinical manifestations and are associated with residual levels of Hex A enzyme activity. We identified a 1422 G-->C (amino acid W474C) substitution in the first position of exon 13 of HEXA of a non-Jewish proband who manifested a subacute variant of G(M2) gangliosidosis. On the second maternally inherited allele, we identified the common infantile disease-causing 4-bp insertion, +TATC 1278, in exon 11. Pulse-chase analysis using proband fibroblasts revealed that the W474C-containing alpha-subunit precursor was normally synthesized, but not phosphorylated or secreted, and the mature lysosomal alpha-subunit was not detected. When the W474C-containing alpha-subunit was transiently co-expressed with the beta-subunit to produce Hex A (alphabeta) in COS-7 cells, the mature alpha-subunit was present, but its level was much lower than that from normal alpha-subunit transfections, although higher than in those cells transfected with an alpha-subunit associated with infantile TSD. Furthermore, the precursor level of the W474C alpha-subunit was found to accumulate in comparison to the normal alpha-subunit precursor levels. We conclude that the 1422 G-->C mutation is the cause of Hex A enzyme deficiency in the proband. The resulting W474C substitution clearly interferes with alpha-subunit processing, but because the base substitution falls at the first position of exon 13, aberrant splicing may also contribute to Hex A deficiency in this proband.

Benign HEXA mutations, C739T(R247W) and C745T(R249W), cause beta-hexosaminidase A pseudodeficiency by reducing the alpha-subunit protein levels.

Two benign mutations, C739T(R247W) and C745T(R249W), in the alpha-subunit of beta-hexosaminidase A (Hex A) have been found in all but one of the currently identified Hex A-pseudodeficient subjects. To confirm the relationship of the benign mutations and Hex A pseudodeficiency and to determine how the benign mutations reduce Hex A activity, we transiently expressed each of the benign mutations, and other mutations associated with infantile, juvenile, and adult onset forms of GM2 gangliosidosis, as Hex S (alphaalpha) and Hex A (alphabeta) in COS-7 cells. The benign mutations decreased the expressed Hex A and Hex S activity toward the synthetic substrate 4-methylumbelliferyl-6-sulfo-beta-N-acetylglucosaminide (4-MUGS) by 60-80%, indicating that they are the primary cause of Hex A pseudodeficiency. Western blot analysis showed that the benign mutations decreased the enzymatic activity by reducing the alpha-subunit protein level. No change in heat sensitivity, catalytic activity, or the substrate specificity to the synthetic substrates, 4-methylumbelliferyl-beta-N-acetylglucosaminide or 4-methylumbelliferyl-6-sulfo-beta-N-acetylglucosaminide, was detected. The effects of the benign mutations on Hex A were further analyzed in fibroblasts, and during transient expression, using pulse-chase metabolic labeling. These studies showed that the benign mutations reduced the alpha-subunit protein by affecting its stability in vivo, not by affecting the processing of the alpha-subunit, i.e. phosphorylation, targeting, or secretion. Our studies also demonstrated that these benign mutations could be readily differentiated from disease-causing mutations using a transient expression system.

Motor function analysis of myelin mutant mice using a rotarod.

We have examined motor control in normal and shiverer mutant mice using the rotarod assay, a forced motor activity which tests for balance and co-ordination. Shiverer mice carry a deletion of the myelin basic protein (MBP) gene, resulting in CNS dysmyelination and characteristic motor dysfunction. Homozygous mutant mice had a significant increase in cumulative falls from the rotarod relative to heterozygous mice. Non-acclimated animals of both genotypes showed progressive improvement in performance when tested on successive days. The rotarod test also discriminated shiverer mutants from animals that received gene therapy intervention. Shiverer animals carrying an MBP transgene showed gene-dosage-dependent improvements in motor function, and mutants which received thalamic transplants of wild type oligodendrocyte precursor cells showed improvement relative to sham operated and non-transplanted controls. Thus the rotarod is a sensitive measure of motor function in hypomyelinated mice, and may be useful for assessing the results of experimental manipulations including transgenic gene therapy and cell transplantation.