Evidence for a Role of the Transcriptional Regulator Maid in Tumorigenesis and Aging

Maid is a helix-loop-helix protein that is involved in cell proliferation. In order to further elucidate its physiological functions, we studied Maid activity in two small fish model systems. We found that Maid expression was greatest in zebrafish liver and that it increased following partial hepatectomy. Maid levels were also high in hepatic preneoplastic foci induced by treatment of zebrafish with diethylnitrosamine (DEN), but low in hepatocellular carcinomas (HCC), mixed tumors, and cholangiocarcinomas developing in these animals. In DEN-treated transgenic medaka overexpressing Maid, hepatic BrdU uptake and proliferation were reduced. After successive breedings, Maid transgenic medaka exhibited decreased movement and a higher incidence of abnormal spine curvature, possibly due to the senescence of spinal cord cells. Taken together, our results suggest that Maid levels can influence the progression of liver cancer. In conclusion, we found that Maid is important regulator of hepatocarconogenesis and aging.


Introduction
Maid was first isolated from mouse embryos and showed that it has Helix-loop-helix (HLH) structure [1]. Maid is also known as CCNDBP1 (Cyclin D1 binding protein), GCIP (Grap2 and cyclin D-interacting protein) and DIP1 (D type cyclin interacting protein 1), and plays a putative role in the inhibition of E2F-1 transcription factor activity [2][3][4]. We previously demonstrated that Maid is a useful marker for human hepatocarcinogenesis since its expression is high in hepatic preneoplastic foci and decreases as cells become progressively less differentiated [5]. Ma et al. examined transgenic mice overexpressing Maid and found that there were no obvious changes in liver development or liver regeneration; however, these mice were less susceptible than wild type (WT) animals to chemical hepatocarcinogenesis induced by diethylnitrosamine (DEN) [6]. Sonnenberg et al. generated Maid knockout (KO) mice, and observed that Maid is a tumor suppressor gene because hepatocellular carcinoma (HCC) development was observed [7]. Consistent with this observation, Chen et al. found that Maid expression was lowest in human breast cancers with very poor prognoses [8]. Other work in human cancer cells has indicated that "Ras associated with diabetes" (Rad) can interact with Maid and block its tumor suppressor activity. Furthermore, when Rad was knocked down in a human cancer cell line, Maid translocated to the nucleus and induced cell cycle arrest and senescence [9]. These results suggest that Maid may also be involved in aging.
Despite the above body of work, it has been difficult to dissect the mechanisms of Maid functions in mammals. To gain deeper insight into Maid mechanisms, we examined its biology in two small fish models. Small fish like zebrafish (Danio rerio) and medaka (Oryzias latipes) can therefore be used to model many aspects of tumorigenesis [10]. Indeed, prior research has demonstrated that a variety of cancers, including hepatic malignancies, can be induced by carcinogens in both these species [11,12]. Thus, to investigate in more depth the functions of Maid in cancer and aging, we characterized Maid expression and functions in zebrafish and transgenic medaka expressing Human Homolog of Maid (HHM). Here, we report the results of our initial investigations of Maid biology in these model systems.

Zebrafish and medaka strains
The RIKEN WT (RW) zebrafish strain (Danio rerio) was obtained from the Riken stock center (the support of National Bioresource Project of Japan) and maintained at 28°C with a 14 h light/10 h dark cycle. Zebrafish were fed living brine shrimp and flake food (Kyowa N type N700). The inbred medaka strain (Kyoto-Cab) was used in this study (Furutani-Seiki et al., 2004). The protocol was approved by the Committee on the Ethics of Animal Experiments of the University of Yamaguchi university (Permit Number: 21S08). All surgery for rabbits or zebrafish was performed under sodium pentobarbital anesthesia or tricaine anesthesia, respectively. All efforts were made to minimize suffering during the course of this study. Rabbit and fish were sacrificed by overdose of anesthesia.

Immunohistochemistry
Tissue samples prepared from zebrafish were fixed in 4% paraformaldehyde and sliced into 3 μm sections using a cryostat. Prior to immunohistochemical staining, endogenous peroxidase in fixed tissue slices was blocked by treatment with fresh 0.3% hydrogen peroxidase in methanol for 30 min at 4°C. Blocked samples were incubated with polyclonal rabbit anti-zebrafish Maid and rabbit anti-HHM antibodies (1:5000) overnight at 4°C according to an established protocol [14]. After washing 3 times in PBS, the sections were incubated with biotin-conjugated secondary antibody in PBS for 3 h at 20°C. After 3 additional PBS washes, a peroxidaseanti-peroxidase complex and streptavidin were added and incubation was maintained at 20°C. Positive reactions were developed for 5-10 min using Tris-HCl buffer containing hydrogen peroxidase and 3,3 0 -diaminobenzidine. Normal goat serum (Vector Laboratories, Burlingame, CA, USA) was used as a negative control.

Partial hepatectomy and fin amputation
Partial hepatectomy was performed as previously described [15]. Briefly, fish were anesthetized, the abdomen was resected, and part of the ventrical lobe of the liver was removed using a scalpel. Liver regeneration was monitored for 7 days. For fin amputation, fins were resected with a scalpel at 1/3 of the total distance from the tip. Fin regeneration was monitored for 14 days.

Chemical carcinogenesis
For zebrafish studies, one-year-old zebrafish were maintained in 6 L of water containing 200 ppm diethylnitrosamine (DEN; Sigma Chemical Co, St Louis MO.) for 3 or 8 weeks, with the water changed once per week. Treated fish were then exposed to 75 mg/L 5-bromo-2 0deoxyuridine (BrdU) solution (Sigma Chemical Co, St Louis MO.) for two days. Fish were sacrificed and cell proliferation analyzed by immunohistochemistry. Preneoplastic foci and tumor types were identified by their size and pattern of HE staining under a light microscope. For HHM transgenic medaka studies (see below), fish were reared in 6 L of water containing 100 ppm DEN for 3 weeks, during which the water was changed weekly. Treated fish were then exposed to 75 mg/L BrdU for 2 days as described above [16]. Fish were sacrificed and cell proliferation analyzed as above. Homozygous, heterozygous and control HHM transgenic medaka were all reared in the same aquarium during DEN treatment.

Generation of HHM transgenic medaka
For HHM expression, a pcDNA3.1 vector was constructed that contained: (1) the HHM coding domain placed under the control of the promoter of the red sea bream β-actin gene, and (2) GFP under the control of a separate red sea bream β-actin promoter.
To establish transgenic medaka strains that ubiquitously expressed Maid, 10 ng/μl circular DNA containing the HHM cDNA under the control of the red sea bream β-actin promoter was injected into one-cell stage embryos of the Kyoto-Cab inbred strain. The circular DNA was prepared using a Qiagen EndoFree plasmid Maxiprep kit (Qiagen, USA). EGFP-positive embryos were selected by fluorescence microscopy, allowed to mature, and bred to WT fish of the Kyoto-Cab inbred strain at 28°C. To identify HHM transgenic fish, livers were isolated from GFP-positive individuals and HHM expression was determined by Western blotting. Representative lines (31 and 52) were selected for experiments. Fish heterozygous for the HHM transgene and non-transgenic siblings were maintained as controls. Transgenic medaka and siblings born on the same day were reared in the same aquarium and used in the same experiment. For chemical carcinogenesis, HHM transgenic medaka were treated with DEN as described [14]. Evaluation of movement was performed with the chronobiology kit for fish.

SA-β-gal staining
Tissue slices (1 mm) were cut from the area of the anus in medaka and stained for 12 hrs using a SA-β-gal staining kit (Biovision) for visualization of senescent cells.

Statistical analyses
All data are expressed as the mean ± S.D. unless otherwise indicated. One way ANOVA followed by the Dunnett post hoc multiple comparison test was performed to assess the statistical significance of differences in the RT-PCR analysis of Maid mRNA expression after PH and fin amputation, and the quantitation of the BrdU labeling results. The Student's t-test was performed to assess the results of the real-time RT-PCR analysis and to compare the body lengths of transgenic medaka. P values less than 0.05 were considered to be significant. Mann-Whitney U test and Student's t-test were performed to assess the statistical significance of differences in neoplastic and tumors between group1 and group2.

cDNA sequence and amino acid sequence comparison
The full length cDNA contained 1210 bp and encoded a 344 amino acid protein that contained putative leucine zipper and HLH domains (S1 Fig). Maid is present in vertebrates, including fish such as the puffer fish and stickleback (S2 Fig). The homology of ZHM at the amino acid level is 38% to humans, 48% to chickens, 43% to Xenopus, 48% to tetraodontidae and 59% to sticklebacks.

Maid expression in normal adult zebrafish
To initiate our delineation of Maid functions in small fish, we examined levels of Maid protein in various tissues of adult zebrafish using Western blotting and Ponseau-S staining. The liver showed the highest Maid levels, although relatively high expression was also noted in the intestine, kidney, and testis ( Fig 1A). Similarly, immunohistochemical staining demonstrated that the highest expression of Maid protein occurred in the liver of zebrafish larvae at 30 days postfertilization (30 dpf) (Fig 1B), with localization predominantly in the cytoplasm of hepatocytes (Fig 1D left). Compared with larvae, hepatocytes in the livers of adult zebrafish showed lower levels of Maid in both the nucleus and cytoplasm (Fig 1B and 1D middle). Moderate Maid expression was observed in the kidneys and uriniferous tubules ( Fig 1C).
We next analyzed Maid expression during zebrafish embryogenesis. High levels of Maid mRNA expression have been documented in human fetal liver [2], and in situ hybridization studies have detected Maid protein in the liver, central nervous system, and dorsal root ganglia of fetal mice [7]. No previous report, however, has addressed changes in Maid protein levels during development. We therefore monitored Maid protein expression during zebrafish embryogenesis and found that maternal-derived protein was present before fertilization, and that Maid protein levels increased as embryonic development progressed (Fig 2A).
Immunohistochemical staining of larvae immediately after hatching revealed high PCNA and Maid expression in the liver, as expected ( Fig 2B). Because these results suggested that Maid might play an important role during embryogenesis, we examined the effect of inhibiting Maid expression during zebrafish development using a specific morpholino antisense oligonucleotide [17]. We confirmed that morpholino injection into one-cell embryos resulted in inhibition of Maid protein production. Nevertheless, the effect of Maid protein knockdown was significant at 16 hours post-fertilization (hpf) and 24 hpf (Fig 2C), no significant changes in larval morphology or time of hatching were observed. Interestingly, analysis of gene expression using real-time PCR revealed that cyclin D1 mRNA was significantly elevated in Maid morpholinoknockdown embryos at 16 and 24 hpf (Fig 2D).

Maid expression during tissue regeneration after partial hepatectomy or fin amputation
The high levels of Maid expression in zebrafish liver led us to hypothesize that Maid might play a critical role in this organ. We therefore examined Maid expression during liver regeneration in WT zebrafish subjected to PH. We found that the resected livers regenerated to their original size in about 7 days ( Fig 3A). Significantly increased amounts of Maid mRNA were present in the liver on day 1 after resection, but these had returned to their pre-resection levels by day 7 (Fig 3B). Howerver no statistical changes were not observed in Westernblotting. We also studied Maid expression during fin regeneration, which is the most frequently examined zebrafish tissue regeneration model [15]. However, no significant changes in Maid levels in mRNA and protein were observed in the regenerating fin at any time point studied ( Fig 3C). These results are consistent with the notion that Maid function is of particular significance for the proliferation of cells during regeneration in the liver but not in the fin.

Maid expression during DEN-induced liver carcinogenesis
To determine the role of Maid during carcinogenesis in small fish, we treated zebrafish with DEN to induce liver cancer. We studied both a short-term group of fish (n = 46) that were reared in fresh water for 5 weeks after 3 weeks of immersion in water containing 100 ppm DEN (Group 1), and a longer-term group (n = 50) that were reared in fresh water for 12 weeks after 8 weeks of immersion in water containing 100 ppm DEN (Group 2). A comparison of these two groups at their endpoints revealed that Group 2 contained more individuals with distended abdomens (Fig 4A, top). When these fish were autopsied, they had extremely swollen livers (Fig 4A, bottom). Western blotting revealed significant decreased expression of Maid in whole swollen livers from Group 2 fish treated with DEN for 8 weeks compared to normal livers ( Fig 4B). In addition, the proliferation marker PCNA was virtually absent from normal livers but elevated in livers from fish treated with DEN for 8 weeks.
We then examined Maid expression in specific tumor types. Most preneoplastic foci positive for both PCNA and BrdU exhibited elevated expression of Maid ( Fig 5A). Moreover, Maid had appeared to translocate to the hepatocyte nucleus (Fig 1D right). In contrast in HCC, which is a tumor type associated with compression of surrounding tissue and a total loss of normal tubular architecture, we observed considerable nuclear localization of PCNA but Maid expression was reduced compared to normal liver ( Fig 5B). Maid protein was also lower in cholangiocarcinomas with developed ductal structures (Fig 5C), and in mixed tumors in which biliary epithelial cells formed small ductules with irregular cords of small hepatocytes ( Fig 5D). These data are consistent with the proposed tumor-suppressive role of Maid in the liver.
Group 1 contained 2 fish (4.3%) with adenoma/HCC. Group 2 contained 15 with adenoma/ HCC (30%), 4 with cholangiocarcinoma (6%), and 13 with mixed tumors (26%) ( Table 1). Neither of the 2 adenoma/HCCs isolated from Group 1 exhibited any change in Maid expression. Preneoplastic foci and vacuolated focus observed in Group1 and Group2 showed no change in Maid expression (Table 2). Maid expression pattern in mixed tumor compared to that in preneoplastic foci was significantly different ( Table 2). Maid expression pattern in adenoma/HCC compared to that in preneoplastic foci showed a tendency toward decreased expression but did not reach statistical significance (p-value = 0.06). These results bolster our conclusion that Maid functions as a moderate tumor suppressor in zebrafish liver.

Decreased liver cell proliferation in DEN-treated Maid transgenic medaka
Medaka have long been used for cancer research, and many mutants are available. We generated transgenic medaka that ubiquitously overexpressed HHM using the sea bream β-actin promoter driving both HHM and EGFP expression. Among the several transgenic strains generated, line 52 exhibited the highest HHM expression, while lines 8, 22 and 31 exhibited lower HHM protein levels (Fig 6A). Lines 52 and 31 were chosen for further study. Sibling embryos that were homozygous for two copies of the HHM transgene [HHM Tg (+/+)], or heterozygous for one copy of the transgene [HHM Tg (+/-)], or non-transgenic siblings [HHM Tg (-/-)] were examined using the method of Brennan et al., who previously reported that transient chemically-induced hepatocyte proliferation in medaka can be assessed using BrdU incorporation [16]. We detected an increase in BrdU positive cells in line 52 and line 31 HHM Tg medaka at 7 days post-DEN administration. We then used BrdU incorporation and immunohistochemistry to detect changes in liver cell proliferation in HHM Tg medaka subjected to DEN exposure for 3 weeks (Fig 6B). The BrdU labeling index was significantly lower in line 52 and line 31 HHM Tg medaka than in their respective control non-transgenic HHM Tg siblings (Fig 6C). Thus, DEN-treated medaka overexpressing Maid proliferate less vigorously than DEN-treated WT medaka, suggesting that Maid also has an anti-tumor effect in this species.

HHM transgenic medaka show delayed growth, decreased daily movement and a hunched back
We noticed that untreated HHM transgenic medaka allowed to reach maturity tended to have a shorter body length than non-transgenic siblings. To confirm this phenotype, siblings born on the same day were reared in a single aquarium and body length was formally measured at one, two and three months after fertilization. Line 52 HHM Tg medaka were significantly smaller in body length than their non-transgenicsiblings at 2 months and 3 months after birth ( Fig 7A). HHM expression was still seen in 1 year old transgenic lines (S3 Fig). No other  obvious differences from WT medaka were observed in either transgenic line up to the age of 10 months. However, when HHM Tg medaka were examined at approx. 12 months of age, many line 52 HHM Tg medaka exhibited a bent "hunched back" phenotype that was rarely seen in non-transgenic siblings of the same age (Fig 7B). This phenotype first appeared around 10 months after birth in both line 52 and line 31 HHM Tg medaka, and was observed in about 80% of line 52 HHM Tg fish at one year after birth. Furthermore, we evaluated movement of Tg medaka with chronobiology kit, and found that line 52 HHM Tg medaka showed decreased movement (Fig 7C). In hematoxylin and eosin-stained sections of the trunks of these fish, there were no obvious morphological changes (such as atrophy) to the musculature. However, we did observe eosinophilic deposits in the spinal cord cells of both lines of HHM Tg medaka that were absent from the spinal cord cells of non-transgenic siblings (S4 Fig), implicating the degeneration of these cells as a possible cause of the spinal abnormality. Furthermore, senescence-associated β-galactosidase staining in the spinal cord cells of HHM transgenic medaka was stronger than in non-transgenic siblings (Fig 7D), suggesting that overexpression of Maid accelerates aging in the spinal cord.

Expression of Maid in a zebrafish liver cell line
To study effects of Maid overexpression in cells, analysis was run using liver-derived cultured cell lines, ZFL. We made ZFL harboring Maid-GFP overexpression vector. Maid-GFP fusion protein was present mainly in the cytoplasm, whereas GFP alone was distributed in both the nucleus and cytoplasm. Studying the proliferative capacity of Maid-GFP stable transformants revealed a slight growth inhibition compared to cells expressing GFP alone (S5 Fig). Furthermore, we performed microarray analysis to investigate gene expression in Maid overexpressing cells. Some interesting changes in up-regulated factors were determined in microarray analysis of ZFL overexpressing Maid. (S1 Table). We focused on TGF-beta as up-regulated factors and showed knowledge-based interaction network of TGF-beta targets after Maid overexpression in ZFL (S6 Fig).

Discussion
In this study, we took advantage of two small fish models to extend the previously known physiological functions of Maid. Our amino acid sequence analysis revealed that Maid's leucine zipper domain, which participates in protein-protein interactions, is highly conserved in zebrafish (S1 and S2 Figs). In addition, although some previous studies have described the abundance of Maid mRNA in specific tissues, our report is the first to investigate tissue-specific protein levels of this transcription factor. Upon examination of Maid protein in various zebrafish organs, we found that the highest levels of this molecule occur in the liver. Previous reports have shown that, upon translocation to the mammalian cell nucleus, Maid appears to exert cytostatic effects via interaction with molecules that are involved in cellular proliferation [18][19][20]. We observed that Maid was localized mainly in the cytoplasm of zebrafish hepatocytes, perhaps reflecting the regenerative potential of this organ. We speculate that Maid's inhibitory effects on proliferation may be initially restrained by its cytoplasmic localization in order to permit hepatocyte proliferation during regeneration following liver damage, but that Maid can rapidly shuttle to the nucleus when needed to later prevent excessive proliferation. However, we acknowledge that Maid interacts with many other molecules in the nucleus and cytoplasm, and may therefore control cellular functions other than proliferation.
To date, there have been no reports on patterns of Maid protein expression during embryogenesis. Since small fish are highly suitable for vertebrate developmental analysis, we used zebrafish for this purpose. We found that unfertilized zebrafish eggs contain maternal-derived Maid protein, and that Maid levels increase following fertilization and development. The transient but robust reduction in Maid protein by knock-down had no effects, suggesting that continuous expression of Maid is dispensable for normal development. Although we saw no obvious defects in zebrafish development following Maid knock-down, we did observe increased expression of cyclin D1 at 16 dpf and 24 dpf. This result is consistent with a previous report in which overexpression of GCIP/Maid suppressed cyclin D1 promoter activity, whereas GCIP/Maid knockdown upregulated cyclin D1 expression [3]. To fully understand the effects of loss of Maid in zebrafish, a genetic knock-out study rather than morpholino knock-down will be required.
The mechanisms responsible for inhibiting hepatocyte proliferation for completing liver regeneration remain elusive. One candidate mechanism to regulate this process is the cytokine TGF-β because TGF-β1 mRNA increases in the first 3-4 h after PH and plateaus after 3 days [21]. We have previously reported that Maid mRNA expression increases transiently after PH in rats [2]. Consistent with this finding, Maid mRNA had increased by day 1 after PH in zebrafish, with a return to normal levels after 7 days. Although Maid mRNA had increased at day1 after PH, there were no statistical significances in Maid protein expression level. We speculate that sensitivity of RT-PCR for change of Maid expression is better than that of protein. We also speculate that turnover rate of Maid was increased, because Maid is known to be degraded by proteasome system quickly [20]. Maid is a synexpression group-restricted regulator of TGF-β [22]. It is therefore possible that the increase in Maid induced by PH suppresses TGF-β signaling and causes it to plateau, decelerating regeneration. Thus, while the cytostatic activity of Maid may play a significant role in the suppression of liver regeneration, the mechanisms involved are likely distinct from those mediated by p21.
With respect to fin regeneration following amputation, no increase in Maid mRNA expression was observed during this process, perhaps reflecting the distinctly different modes of regeneration occurring in these two tissues. While simple duplication of mature hepatocytes in the remaining liver compensates for the lost tissue in post-PH liver, fin regeneration requires mature fin cells to de-differentiate and form blastemas [23].
In addition to its proposed role during liver regeneration, Maid has been considered a putative tumor suppressor gene. The onset of tumorigenesis is suppressed in transgenic mice overexpressing Maid [6], and liver cancer occurs more frequently in Maid knockout mice than in WT controls [7]. In our short-term in vivo study of zebrafish, 4/11 (36%) of the preneoplastic foci induced by DEN administration showed elevated Maid protein. It is possible that this increase in Maid expression is an antiproliferative response to DEN treatment that prevents full-blown tumorigenesis. However, the fact that Maid expression was unchanged in 27% of preneoplastic foci and decreased in 36% of them suggests that Maid levels may be directly related to the degree of malignancy and differentiation of a transformed cell. Similar correlations have been found in the case of other tumor suppressors.
Maid is an HLH transcriptional regulator, a class of factor for which subcellular localization is important. In line with this requirement, we found Maid to be abundant in the cytoplasm of larval hepatocytes immediately after hatching, a time when active proliferation takes place. Maid was also abundant in the cytoplasm of hepatocytes of adult WT zebrafish. After DEN administration, however, Maid appeared to translocate to the hepatocyte nucleus, presumably to participate in growth-inhibitory signaling. Consistent with the importance of the nuclear localization of Maid for its tumor suppressor activity, it has recently been reported in human cancer cells that RRAD (ras associated with diabetes) antagonizes Maid's anti-tumor role by facilitating Maid export from the nucleus to the cytoplasm. Furthermore, RRAD knockdown in human cancer cells results in cell cycle arrest and premature senescence associated with nuclear translocation of Maid [9].
Our study also examined the cytostatic properties of Maid in detail. To this end, we generated transgenic medaka overexpressing HHM and found that high levels of this protein suppressed the hepatocyte proliferation that normally follows DEN treatment. Since HHM is normally localized in the cytoplasm, it does not exhibit a strong cytostatic effect. However, we speculate that, upon potentially tumorigenic stresses such as DEN treatment, the nuclear translocation of HHM is stimulated and its cytostatic effects are manifested.
Although no changes to the liver or other obvious morphological changes were observed during development, HHM transgenic medaka had shorter body lengths than non-transgenic siblings and showed delayed growth. Systemic Maid overexpression is supposed to inhibit cell growth, but we did not detect any significant differences in the expression of cell cycle-related genes such as cyclin D1. This suggests that most of the HHM protein could be localized in the hepatocyte cytoplasm and could not exert its nuclear function. Although no 10-month-old HHM transgenic medaka exhibited an obvious senescence-like phenotype in any tissue analyzed, some of these fish developed a hunched back phenotype sometimes seen in very old WT medaka. While no changes to the musculature in the spinal region of HHM transgenic medaka were detected, deposition of intracellular granules was observed in the spinal cord, suggesting that neurodegeneration may have contributed to the bending of the trunk.
Maid has been previously linked to senescence in human cancer cell line [9]. We found that staining with SA-β-galactosidase, which is used as a senescence marker, was more intense in the spinal cords of HHM transgenic medaka, implying that senescence had accelerated. This is consistent with the report that RRAD knockdown in human cancer cells results in nuclear translocation of Maid and premature senescence [9]. We examined gene expression in ZFL cell lines by microarray analysis, and found that some important upstream regulators (S1 Table). Estrogen plays a very important role in a variety of biological aspects. It is reported that 17beta-E2 might relieve H2O2-induced mitochondrial damage through estrogen receptor and delay the vascular endothelial cell senescence. It is interesting to find TGF-beta as a upstream regulator, because it is related to senescence, and Maid is a synexpression group-restricted regulator of TGF-beta signalling. Furthermore, Maid can participate in a protein complex with SirT6 [24], which is a histone H3 deacetylase involved in maintaining telomeres and chromosomes, repairing DNA, and controlling metabolism. SirT6 knockout mice die one month after birth due to premature aging-like symptoms, such as lordokyphosis. Thus, Maid may exert some of its senescence-promoting effects via functional inhibition of SirT6, a possibility currently under examination.
In conclusion, our work has demonstrated that Maid has cytostatic activity in non-mammalian vertebrates, and that nuclear translocation is likely required for this effect. A greater mechanistic understanding of this process may point toward potential strategies for activating Maid in order to inhibit tumor growth.