Neurons in Alzheimer disease emerge from senescence.

A number of cell cycle markers are associated with the selective neuronal pathology found in Alzheimer disease. However, the significance of such cell cycle markers is clouded by duplicity of function in that many such proteins are also involved in apoptosis and/or DNA repair following oxidative damage. To clarify whether or not neurons in Alzheimer disease do in fact emerge from a quiescent status, with subsequent entry into the G1 phase of the cell cycle, in this study we focused on a family of MORF4-related proteins that are associated with emergence from senescence. Our results show that many neurons in vulnerable regions of Alzheimer disease brain, but not in control brain, have increased MORF4-related proteins indicating re-entry into the cell cycle. Immunoblot analysis showed a specific disease-related increase in a 52 kDa protein that is likely the human homologue of the MORF4-related transcription factor. The novel localization of such a transcriptional activating protein to selectively vulnerable neurons in Alzheimer disease provides compelling evidence for mitotic re-entry as part of the pathogenesis of neuronal dysfunction and death in Alzheimer disease.

MRG15 activates the B-myb promoter through formation of a nuclear complex with the retinoblastoma protein and the novel protein PAM14.

The MORF4-Related Gene on chromosome 15 (MRG15) is a member of a novel family of genes originally identified in studies to reveal cell senescence-inducing factors. MRG15 contains several predicted protein motifs, including a nuclear localization signal, a helix-loop-helix region, a leucine zipper, and a chromodomain. These motifs are commonly associated with transcription factors, suggesting that MRG15 may likewise function as a transcriptional regulator. To examine the potential function(s) of MRG15, we sought to identify cellular factors associated with this MRG family member. In this regard, we have found that both the retinoblastoma tumor suppressor (Rb) and a novel nuclear protein PAM14 (Protein Associated with MRG, 14 kDa) specifically associate with MRG15. We have further demonstrated that these interactions require the helix-loop-helix and leucine zipper domains of MRG15. Interestingly we have found all three proteins present in a multiprotein complex, suggesting that at least some of their functions may be interdependent. Although the functions of PAM14 have yet to be elucidated, Rb has several well characterized activities, including repression of E2F-activated promoters such as that of B-myb. Significantly we have demonstrated that MRG15 blocks the Rb-induced repression of this promoter, leading to B-myb promoter activation. Collectively these results suggest that MRG15 regulates transcription through interactions with a cellular protein complex containing Rb and PAM14.

Conservation of the MORF4 related gene family: identification of a new chromo domain subfamily and novel protein motif.

The seven member, human MORF4 related gene (MRG) family was recently identified based on the ability of Mortality factor on chromosome 4 (MORF4) to induce replicative senescence in immortal cell lines assigned to complementation group B (Bertram et al., 1999. Mol. Cell Biol. 19, 1479-1485). Initial computer based similarity searches identified human retinoblastoma binding protein 1 (RBP-1), Drosophila melanogaster male specific lethal-3 (Msl-3), S. pombe altered polarity-13 (Alp13) and S. cerevisiae Eaf3p, a component of the yeast NuA4 HAT complex (Galarneau et al., 2000. Mol. Cell 5, 927-937), as having similarity to the human MRG protein family. This suggested that the MRG family might be found in multiple species, and analysis of other homologs would provide functional and evolutionary insights into this gene family. Here, we report that MRG family members are present in twenty-three species based on molecular assays and sequence similarity searches. The new family members were divided into two groups based on similarity to the predominant human MRG family members, MRG15 and MRGX. The family members similar to MRG15 define a new, highly conserved subsection of the chromo domain superfamily. Additionally, conservation in the C-terminal two thirds of all the MRG family members and the Drosophila and human MSL-3 proteins defines a new protein domain, the MRG domain. These results indicate a highly conserved role for the MRG family in transcriptional regulation via chromatin remodeling by histone acetylation.

Identification of genes involved in cell senescence and immortalization: potential implications for tissue ageing.

The limited proliferative potential of normal cells in culture, cell replicative senescence, is an accepted model for ageing at the cellular level. Tumour-derived, or viral- or carcinogen-transformed cells have escaped senescence and proliferate without control (immortal). We and others have found that fusion of normal with immortal human cells yields hybrids that have regained growth control and cease division. This demonstrates that the phenotype of replicative senescence is dominant and that cells immortalize because of defects in senescence-related genes. We exploited the recessive nature of immortality and by fusing different immortal cell lines with each other identified four complementation groups for indefinite division. Immortal parental cell lines with similar senescence gene defects when fused yielded hybrids with unlimited division potential and were assigned to the same complementation group. Fusion of immortal cell lines with different gene defects resulted in complementation in the hybrids, which had limited division capability. These parental cell lines were assigned to different complementation groups. Using microcell-mediated chromosome transfer, we then demonstrated that introduction of a normal human chromosome 4 induced senescence only in immortal cell lines assigned to complementation group B. We have now cloned the gene on chromosome 4, MORF4 (mortality factor on chromosome 4). It is a member of a family of seven genes and only MORF4 and the MORF-related genes MRG15 and MRGX are expressed. The predicted protein motifs strongly suggest this is a novel family of transcription factors. We have identified interacting proteins, some of which are also novel. These genes have the potential to modulate expression of a large number of genes by chromatin remodelling. They, therefore, also have the potential to affect tissue function due to changes in expression activity during ageing.

Characterization of a novel zinc finger gene with increased expression in nondividing normal human cells.

We report here the cloning and characterization of a novel KRAB zinc finger gene, ZFQR, which has eight tandemly repeated zinc fingers, a complete KRAB box at the N-terminal region, and a unique C-terminal sequence. It is expressed in a variety of human tissues, and mRNA levels are upregulated in nondividing senescent and quiescent human fibroblasts. Overexpression of the protein in quiescent cells stimulated with serum growth factors results in inhibition of entry into the cell cycle. The latter activity is lost when the N-terminal KRAB domain is deleted. The KRAB domain is also required for the transcriptional repression activity of ZFQR and in maintaining association of the protein with the nuclear matrix. The gene has been mapped to human chromosome 19q13.4. The association of ZFQR with the nuclear matrix, transcriptional repression activity, increased expression in senescent and quiescent cells, and the ability to inhibit quiescent cells stimulated with growth factors from entering the cell cycle suggests a role for ZFQR in the maintenance of the nondividing state of normal human cells.

Identification of an alternatively spliced form of the Tat interactive protein (Tip60), Tip60(beta).

Tip60 was originally isolated as a Tat interactive protein. It was subsequently shown that Tip60 had histone acetyltransferase (HAT) activity. In studies to understand gene-expression regulation that might involve HAT activity, we PCR-amplified Tip60 from a human heart marathon-ready cDNA library. As a result, we identified an alternatively spliced form of Tip60, Tip60beta (we refer to the previously cloned Tip60 as Tip60alpha). Tip60beta cDNA is slightly smaller than Tip60alpha, and sequencing indicates that there is a deletion of 156 bp in the coding region of the gene. The predicted Tip60beta protein therefore lacks 52 amino acids when compared with Tip60alpha. The Tip60alpha gene is encoded by 14 exons, and Tip60beta is an alternatively spliced form resulting from the exclusion of exon 5 during the splicing process. Exon 5 encodes a proline-rich region that is known to be important for protein-protein interaction. Tip60beta is expressed in a variety of human tissues and cell lines, and the protein is present in both the nucleus and cytoplasm in contrast to Tip60alpha, which is entirely nuclear. The results suggest that Tip60beta may have functions additional to those of Tip60alpha in cells and tissues.

Extramitochondrial localization of mortalin/mthsp70/PBP74/GRP75.

Subcellular fractionation and immunofluorescence microscopy were used to identify the specific sites of intracellular residence of mortalin, also called a mitochondrial homologue of the hsp70 family, in immortal human cell lines previously assigned to four distinct complementation groups (A-D) for indefinite cell division. In addition to the mitochondria it was seen in the endoplasmic reticulum (ER) fractions of all the cell lines analyzed. Interestingly, three of the group A cells lines (EJ, GM639, and HT1080), in addition to the mitochondria and ER, exhibited cytosolically (extra-organelle) localized pool of mortalin. These findings demonstrate that mortalin is not present exclusively in mitochondria. Its residence in different organelles may be the basis of differential distribution observed previously in different human cell lines.

Genetic approaches to the study of replicative senescence.

Genetic analyses of replicative senescence have revealed the dominance of the senescent phenotype since whole cell fusion of normal with immortal cells yields hybrids having limited division potential. We exploited the recessive nature of immortality by fusing different immortal human cell lines with each other and identified four complementation groups for indefinite division. This allowed for a focussed approach involving microcell mediated chromosome transfer that led to the implication of chromosomes 1, 4 and 7 as loci for cell senescence genes. More recently we have cloned the gene on chromosome 4, MORF 4. It is a member of a family of genes with motifs suggestive of transcriptional regulators. Characterization of this novel gene family should lend further insights into the phenomenon of replicative cell senescence.

Genetic and functional analyses exclude mortality factor 4 (MORF4) as a keratinocyte senescence gene.

Approximately 50% of immortal human keratinocyte lines show loss of heterozygosity of chromosome region 4q33-q34, and the reintroduction of chromosome 4 into one such line, BICR 6, causes proliferation arrest and features of replicative senescence. Recently, a candidate gene, mortality factor 4 (MORF4), was identified in this region and sequenced in 21 immortal keratinocyte lines. There were no mutations or deletions, and two of the seven lines that showed loss of heterozygosity at 4q33-q34 were heterozygous for MORF4 itself. Furthermore, the transfer of a chromosomal segment containing the entire MORF4 gene did not mimic the senescence effect of chromosome 4 in BICR 6. These results suggest that the inactivation of MORF4 is not required for human keratinocyte immortality.

Cloning and characterization of CRF, a novel C1q-related factor, expressed in areas of the brain involved in motor function.

We have isolated and characterized a novel cDNA, C1q-Related Factor (CRF), that is predicted to encode a 258 amino acid polypeptide with a hydrophobic signal sequence, a collagenous region, and a globular domain at the carboxy terminus that shares homology to the C1q signature domain. Human CRF transcript is expressed at highest levels in the brain, particularly in the brainstem. In situ hybridization to mouse brain sections demonstrated that CRF transcripts are most abundant in areas of the nervous system involved in motor function, such as the Purkinje cells of the cerebellum, the accessory olivary nucleus, the pons and the red nucleus. The mouse CRF homolog is highly similar to the human gene at both the nucleotide and protein level, suggesting an important conserved role for this protein.

Identification of a gene that reverses the immortal phenotype of a subset of cells and is a member of a novel family of transcription factor-like genes.

Based on the dominance of cellular senescence over immortality, immortal human cell lines have been assigned to four complementation groups for indefinite division. Human chromosomes carrying senescence genes have been identified, including chromosome 4. We report the cloning and identification of a gene, mortality factor 4 (MORF 4), which induces a senescent-like phenotype in immortal cell lines assigned to complementation group B with concomitant changes in two markers for senescence. MORF 4 is a member of a novel family of genes with transcription factor-like motifs. We present here the sequences of the seven family members, their chromosomal locations, and a partial characterization of the three members that are expressed. Elucidation of the mechanism of action of these genes should enhance our understanding of growth regulation and cellular aging.

Suppression of transformed phenotype and tumorigenicity after transfer of chromosome 4 into U251 human glioma cells.

The development of primary human brain tumors, particularly glioblastoma multiforme (GBM), has been associated with a number of molecular and chromosomal abnormalities. In this study, a novel tumor suppressor locus was identified and localized after the transfer of a human chromosome 4 into U251 human GBM cells. Hybrid clones containing a transferred neomycin-resistance tagged chromosome 4 revealed an inability to form tumors in nude mice and a greatly decreased efficiency of soft agarose colony formation. As a control, clones containing a transferred chromosome 2 were generated, and these retained the tumorigenic phenotype of the parental U251 cells. The presence of the transferred chromosomes was demonstrated by gain of polymorphic loci and FISH analyses. Several suppressed hybrid clones were shown to contain spontaneously reduced versions of the transferred chromosome 4. A common region of the fragmented chromosome 4 was retained among these clones that included the epidermal growth factor locus at 4q24-26 and several adjacent markers. The identification of a common fragment in the suppressed clones suggests the presence of a tumor suppressor gene or genes in this region, involved in glioma oncogenesis.

Loss of T-antigen sequences allows SV40-transformed human cells in crisis to acquire a senescent-like phenotype.

Normal human cells transfected with SV40 DNA exhibit an extended proliferative potential compared with controls, but they eventually enter a phase known as "crisis." During crisis, extensive cell death occurs and the cells exhibit some gene expression changes similar to senescent cells. This article presents results which indicate that crisis most likely depends on expression of the viral gene T-antigen. We have obtained a unique subpopulation of cells that have deleted the T-antigen gene and, rather than dying as cells do in crisis, remain viable and exhibit some senescent-like characteristics. We also found that the SV40 promoter is poorly expressed in senescent versus young cells. We hypothesize that decreased activity of the viral promoter may result in decreased expression of T-antigen, which is challenged by over-expression of the cell cycle inhibitors such as p21Sdi1. Conflicting signals to proceed/halt cells cycle progression result in the cell death associated with crisis.

Replicative senescence: implications for in vivo aging and tumor suppression.

Normal cells have limited proliferative potential in culture, a fact that has been the basis of their use as a model for replicative senescence for many years. Recent molecular analyses have identified numerous changes in gene expression that occur as cells become senescent, and the results indicate that multiple levels of control contribute to the irreversible growth arrest. These include repression of growth stimulatory genes, overexpression of growth inhibitory genes, and interference with downstream pathways. Studies with cell types other than fibroblasts will better define the role of cell senescence in the aging process and in tumorigenesis.

Evidence for two senescence loci on human chromosome 1.

Microcell-mediated introduction of a neo-tagged human chromosome 1 (HC-1-neo) into several immortal cell lines has previously been shown to induce growth arrest and phenotypic changes indicative of replicative senescence. Somatic cell hybridization studies have localized senescence activity for immortal hamster 10W-2 cells to a cytogenetically defined region between 1q23 and the q terminus. Previous microcell-mediated chromosome transfer experiments showed that a chromosome 1 with an interstitial q-arm deletion (del-1q) lacks senescence inducing activity for several immortal human cell lines that are sensitive to an intact HC-1-neo. In contrast, our studies reveal that the del-1q chromosome retains activity for 10W-2 cells, indicating that there are at least two senescence genes on human chromosome 1. Sequence-tagged site (STS) content analysis revealed that the q arm of the del-1q chromosome has an interstitial deletion of approximately 63 centimorgans (cM), between the proximal STS marker DIS534 and distal marker DIS412, approximately 1q12 to 1q31. This deletion analysis provides a candidate region for one of the senescence genes on 1q. In addition, because this deletion region extends distally beyond 1q23, it localizes the region containing a second senescence gene to approximately 1q31-qter, between DIS422 and the q terminus. STS content analysis of a panel of 11 10W-2 microcell hybrid clones that escaped senescence identified 2 common regions of loss of 1q material below the distal breakpoint of del-1q. One region is flanked by markers DIS459 and ACTN2, and the second lies between markers WI-4683 and DIS1609, indicating that the distal 1q senescence gene(s) localizes within 1q42-43.

Studies demonstrating the complexity of regulation and action of the growth inhibitory gene SDI1.

The identification of the DNA synthesis inhibitory gene SDI1 by investigators studying cell senescence, tumor suppression, cell cycle control and differentiation suggest a key regulatory role for this gene. To better understand the growth regulatory activity of this gene we proceeded with the experiments described here. The data demonstrate that SDI1 is an important downstream effector of p53, but here we report that it can also cause inhibition of DNA synthesis in several immortal human cell lines, independent of p53 or Rb status. Levels of SDI1 mRNA expression in immortal cells are consistently much lower than that of normal cells, indicating that immortalization is not compatible with high expression of SDI1. These results highlight the complex nature of regulation of this gene and its mode of action.