Upregulation of the gene encoding a cytoplasmic dynein intermediate chain in senescent human cells

Normal human somatic cells, unlike cancer cells, stop dividing after a limited number of cell divisions through the process termed cellular senescence or replicative senescence, which functions as a tumor‐suppressive mechanism and may be related to organismal aging. By means of the cDNA subtractive hybridization, we identified eight genes upregulated during normal chromosome 3‐induced cellular senescence in a human renal cell carcinoma cell line. Among them is the DNCI1 gene encoding an intermediate chain 1 of the cytoplasmic dynein, a microtubule motor that plays a role in chromosome movement and organelle transport. The DNCI1 mRNA was also upregulated during in vitro aging of primary human fibroblasts. In contrast, other components of cytoplasmic dynein showed no significant change in mRNA expression during cellular aging. Cell growth arrest by serum starvation, contact inhibition, or γ‐irradiation did not induce the DNCI1 mRNA, suggesting its specific role in cellular senescence. The DNCI1 gene is on the long arm of chromosome 7 where tumor suppressor genes and a senescence‐inducing gene for a group of immortal cell lines (complementation group D) are mapped. This is the first report that links a component of molecular motor complex to cellular senescence, providing a new insight into molecular mechanisms of cellular senescence. J. Cell. Biochem. 82:415–421, 2001. © 2001 Wiley‐Liss, Inc.

Normal human somatic cells stop dividing after a ®nite number of cell divisions. This process is termed cellular senescence or replicative senescence. In contrast, most cancer cells have an in®nite growth potential, indicating that escape from cellular senescence, or immortalization, is an important step in human carcinogenesis [Chiu and Harley, 1997;Wynford-Thomas, 1999;Horikawa et al., 2000]. Cellular senescence is a complex cellular process regulated by multiple genetic pathways, which re¯ect various intracellular and extracellular signals [Horikawa et al., 2000]. Cell hybrid and chromosome transfer experiments have suggested that immortalization results from functional inactivation of senescence genes Smith, 1983, 1988]. Although a class of senescence genes is suggested to function through the repression of telomerase activity [Horikawa et al., , 2000Tanaka et al., 1998;Cuthbert et al., 1999], molecular mechanisms of cellular senescence are still largely elusive. An approach to better understanding of cellular senescence is to investigate senescence-associated changes in gene expression. Such investigations have found a number of senescence-associated genes, such as cell cycle regulatory genes, growth regulatory genes, and extracellular matrixrelated genes [Cristofalo et al., 1998;Jansen-DuÈ rr, 1998].
We previously reported that microcellmediated transfer of a normal human chromosome 3 into a renal cell carcinoma cell line (RCC23) induced cellular senescence through the repression of telomerase activity and the progressive shortening of telomeric DNA . By means of an ef®cient cDNA subtraction method [suppression subtractive hybridization (SSH)] [Diatchenko et al., 1996], we here identify the genes whose expression is upregulated during the chromosome 3induced cellular senescence. One of those genes encodes a subunit of a microtubule-associated molecular motor complex, providing a novel insight into the molecular mechanism of cellular senescence.

Cells
A renal cell carcinoma cell line, RCC23, was previously established from a non-papillary renal cell carcinoma and expresses telomerase activity ]. Microcellmediated transfer of a single copy of normal human chromosome 3 into RCC23 cells produced a telomerase-negative microcell hybrid (RCC233), which showed progressive shortening of telomeres as a function of cell division numbers ]. RCC233 cells used in this study [clone #3-C in Horikawa et al., 1998] stopped growing at 41 population doublings (PDs) with an expression of the senescence-associated b-galactosidase [Dimri et al., 1995]. A revertant clone (REV) emerged from the senescent RCC233 cells with a loss of the 3p12±p22 region of the transferred chromosome. REV cells showed telomerase activity comparable to parental RCC23 cells, and continued to grow over 100 PDs without any sign of senescence ].
cDNA Subtraction by SSH Cytoplasmic RNA was isolated from RCC23 and RCC233 cells (at 30 PDs) as previously described [Sambrook et al., 1989] and treated with DNase I (Ampli®cation Grade, Life Technologies, Inc., Gaithersburg, MD). Poly(A) RNA was puri®ed from the cytoplasmic RNA by using the Oligotex Direct mRNA Kit (Qiagen, Inc., Chatsworth, CA). To isolate genes which are preferentially or exclusively expressed in RCC233 cells, SSH was performed using the PCR-Select cDNA subtraction kit (Clontech Labs, Inc., Palo Alto, CA) as previously described [Diatchenko et al., 1996]. Starting with 2 mg of poly(A) RNA from RCC233 as a tester and RCC23 as a driver, ®rst-and secondstrand cDNA synthesis, Rsa I digestion of cDNA, ligation of the adapters to the tester cDNA, two steps of subtractive hybridization, and PCR ampli®cation of the subtracted cDNA were carried out according to the manufacturer's instruction. The ®nal PCR products were cloned in pCR2.1 vector (Invitrogen, Corp., San Diego, CA). Differential screening was performed using the 32 P-labeled, subtracted cDNA described above and the 32 P-labeled, reversesubtracted (RCC23 minus RCC233) cDNA similarly obtained as the probes. Plasmid DNA was puri®ed from bacterial colonies which showed preferential hybridization signals with the former probe.

DNA Sequencing and Homology Search
Double-stranded plasmid DNA was sequenced with M13-forward and reverse primers and, if necessary, gene-speci®c internal primers using the dRhodamine terminator cycle sequencing kit (PE Applied Biosystems, Foster City, CA). DNA homology search was performed at the National Center for Biotechnology Information (NCBI) using the basic local alignment search tool (BLAST; http:/ / www.ncbi.nlm.nih.gov/BLAST). Chromosomal localization of the genes was based on the Online Mendelian Inheritance in Man (OMIM; http:/ /www3.ncbi.nlm.nih.gov/Omim/searchmap.html) and/or the GeneMap98 (http:/ / www.ncbi.nlm.nih.gov/genemap98).

Northern Blot and Reverse Transcription (RT)-PCR Analyses
Total cellular RNA was isolated by the RNeasy Mini kit (Qiagen, Inc.) and electrophoresed through a 1.2% agarose±formaldehyde gel. Northern blotting and hybridization were carried out as previously described ].

Successful Application of SSH to the Identi®cation of Differentially Expressed Genes
When 500 bacterial colonies were screened with the subtracted and reverse-subtracted probes (see Materials and Methods), 100 (20%) colonies showed exclusive or preferential hybridization to the former probe. Ten colonies with relatively strong hybridization signal were grown, sequenced, and found to represent eight different genes (Table I). Northern blot analysis con®rmed that all of these eight genes were expressed more abundantly in RCC233 cells than in parental RCC23 cells (data not shown; Fig. 1A). Four genes (plasminogen activator inhibitor-1, extracellular protein S1-5, ®bronectin, and 22-kDa smooth muscle protein) were previously reported to be upregulated during cellular senescence [Thweatt et al., 1992;Kumazaki et al., 1993;Lecka-Czernik et al., 1996;West et al., 1996]. Two genes (growth arrest speci®c protein-6 and follistatin-related protein precursor) were previously linked to cell growth arrest [Schneider et al., 1988;Shibanuma et al., 1993], consistent with their upregulation during cellular senescence. Successful identi®cation of these genes suggests that SSH method worked well in this study and that RCC233 cells are a good cell system to identify senescence-associated genes. One clone showed identity to two sequences of unknown function (GenBank accession numbers: AB033025 and AK000465) that seemed to represent partial cDNA clones. Isolation and detailed characterization of the full-length cDNA will be reported elsewhere. We here direct our attention to the DNCI1 gene encoding an intermediate chain 1 of the cytoplasmic dynein, a microtubule-associated molecular motor complex that plays a role in chromosome movement and intracellular transport [Vallee and Sheetz, 1996;Endow, 1999;Karki and Holzbaur, 1999] but has not been linked to cellular senescence.

DNCI1 mRNA Expression Increases as Cells Age
A 740-bp RsaI±RsaI fragment from 3 0 -region of the DNCI1 cDNA (nucleotides 1792±2531 of GenBank AF063228) [Crackower et al., 1999] was initially isolated by SSH. 5 0 -RACE (rapid ampli®cation of cDNA end) using the primers within this 740-bp region resulted in 2.0-kb fragment containing the entire coding region, which matched the reported DNCI1 sequence. Northern blot analysis (Fig. 1A) showed that RCC233 cells expressed higher levels of DNCI1 mRNA than parental RCC23. The DNCI1 mRNA expression in RCC233 cells increased as a function of PDs (compare lanes 2±4 in Fig. 1A), with a maximum expression at  Tanaka et al., 1998), the amount of DNCI1 mRNA decreased back to an undetectable level (lane 5 in Fig. 1A). RT-PCR analysis also con®rmed a marked increase in DNCI1 mRNA in RCC233 cells (Fig. 1B). These results suggest that induction of the DNCI1 expression is associated with cellular senescence induced by chromosome 3 in RCC23 cells. Cellular aging-associated DNCI1 upregulation was also observed in NHF that were primary cultures from neonatal foreskin successively passaged until they became senescent (Fig. 2) [Vallee and Sheetz, 1996;Endow, 1999;Karki and Holzbaur, 1999]. We examined by RT-PCR analyses whether the cellular senescence-associated upregulation is speci®c to the DNCI1 or generally observed in multiple components. As shown in Figure 3A, we did not ®nd a signi®cant difference in mRNA expression of the other components tested (heavy chain 1, intermediate chain 2, and light chain 1) among RCC23, RCC233, and REV cells. Similarly, mRNA expression of these components showed no signi®cant change throughout the replicative life span of NHF (Fig. 3B). Thus, the cellular senescence-associated mRNA induction is likely to be unique to the DNCI1 among multiple components of cytoplasmic dynein.  , 4 and 6). The control ampli®cation of GAPDH is shown.  Figure 1B. Lanes 2, 4, and 6 are reverse transcriptase-minus controls.

Induction of DNCI1 mRNA is Speci®c to Cellular Senescence
To examine whether or not the upregulation of DNCI1 mRNA is associated with cell growth arrest in general, we induced growth arrest in NHF cells by serum starvation, contact inhibition or g-irradiation. Unlike cellular senescence, none of these conditions resulted in a signi®cant increase in DNCI1 mRNA expression (Fig. 4). This suggests that the induction of DNCI1 mRNA is characteristics of cellular senescence rather than a general feature of cell growth arrest.

DISCUSSION
In this study we identi®ed the DNCI1 gene, which encodes an intermediate chain 1 of the cytoplasmic dynein, as a novel senescenceassociated gene. The DNCI1 upregulation during cellular aging was observed in both epithelial (RCC233) and ®broblastic (NHF) cells. Unlike the DNCI1 gene, expression of genes encoding the other components of cytoplasmic dynein showed no signi®cant change during cellular senescence. Also important is that the DNCI1 upregulation was speci®cally associated with cellular senescence, and not cell growth arrest in general. These ®ndings strongly support a speci®c role of the DNCI1 gene in cellular senescence. This is the ®rst report that links a component of molecular motor complex to cellular senescence, providing a new insight into molecular mechanisms of cellular senescence. Mouse Dnci1 mRNA is expressed most abundantly in brain [Crackower et al., 1999]. By northern blot analysis of normal human tissues (data not shown), we also observed the highest DNCI1 expression in human brain. Human heart and skeletal muscle also expressed a high level of DNCI1 mRNA. It should be noted that a low but signi®cant level of DNCI1 expression was detected in all of the other human tissues and cells examined (i.e., pancreas, kidney, liver, lung, placenta, colon, small intestine, ovary, testis, prostate, thymus, spleen, and mammary epithelial cells). This ®nding suggests a physiological role of the DNCI1 in a wide range of human tissue and cell types.
A precise function of the DNCI1 protein in cellular senescence still remains to be determined. It is unlikely that the DNCI1 is related to telomerase regulation, because the progressive  increase in DNCI1 mRNA expression occurred with no change in telomerase activity (i.e., with telomerase activity kept repressed) during in vitro aging of NHF and RCC233 cells. Regulations of chromosome movement and organelle transport, in which the cytoplasmic dynein is suggested to play important roles [Vallee and Sheetz, 1996;Endow, 1999;Karki and Holzbaur, 1999], may be related to cellular senescence. For instance, altered control of mitotic chromosomes could contribute to permanent cell cycle arrest at cellular senescence, which may be consistent with an aging-related misregulation of mitotic cell division [Ly et al., 2000]. Production of a DNCI1-speci®c antibody and determination of cellular localization of the DNCI1 protein during cellular aging will facilitate better understanding of its biological signi®cance in cellular senescence.
It should be noted that the DNCI1 gene is located on the long arm of chromosome 7 (7q21.3±q22.1, Table I), where putative tumor suppressor genes of various human cancers (e.g., ovarian, breast, and prostate cancers) are mapped [Verma et al., 1999;Zeng et al., 1999;Neville et al., 2001]. In our preliminary examination of seven human cancer cell lines (3 breast and 4 ovarian) for DNCI1 mRNA, no or little expression was detected in a breast cancer cell line MCF-7 and an ovarian cancer cell line BG-1. The chromosome 7q is also supposed to have a senescence-inducing gene whose inactivation is critical to immortality of a group (complementation group D) of human cell lines [Ogata et al., 1995;Nakabayashi et al., 1997]. Of great interest will be whether loss or mutation of the DNCI1 gene plays a causative role in human cell immortalization and caricinogenesis. A potential signi®cance of alternatively spliced forms of DNCI1 [Crackower et al., 1999] also remains to be examined.
Finally, only a small number of clones were the subject of this study. The successful application of SSH shown here suggests that a largescale study to ®nd more differentially expressed genes may lead to the identi®cation of a gene on chromosome 3p that triggered to induce cellular senescence in RCC23 cells.