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The critical role of germinal center-associated nuclear protein in cell biology, immunohematology, and hematolymphoid oncogenesis

  • Author Footnotes
    1 YS and KK contributed equally to this work.
    Yasuhiro Sakai
    Correspondence
    Address correspondence to: Yasuhiro Sakai or Kazuhiko Kuwahara, Department of Diagnostic Pathology, Fujita Health University School of Medicine, 1–98 Dengakugakubo, Kutsukake, Toyoake, Aichi 470–1192, Japan
    Footnotes
    1 YS and KK contributed equally to this work.
    Affiliations
    Department of Diagnostic Pathology, Fujita Health University School of Medicine, Toyoake, Japan
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  • Suchada Phimsen
    Affiliations
    Department of Biochemistry, Faculty of Medical Science, Naresuan University, Phitsanulok, Thailand
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  • Seiji Okada
    Affiliations
    Division of Hematopoiesis, Joint Research Center for Retroviral Infection, Kumamoto University, Kumamoto, Japan
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  • Author Footnotes
    1 YS and KK contributed equally to this work.
    Kazuhiko Kuwahara
    Correspondence
    Address correspondence to: Yasuhiro Sakai or Kazuhiko Kuwahara, Department of Diagnostic Pathology, Fujita Health University School of Medicine, 1–98 Dengakugakubo, Kutsukake, Toyoake, Aichi 470–1192, Japan
    Footnotes
    1 YS and KK contributed equally to this work.
    Affiliations
    Department of Diagnostic Pathology, Fujita Health University School of Medicine, Toyoake, Japan
    Search for articles by this author
  • Author Footnotes
    1 YS and KK contributed equally to this work.
Open ArchivePublished:August 19, 2020DOI:https://doi.org/10.1016/j.exphem.2020.08.007

      Highlights

      • GANP plays multifunctional critical roles in cell biology and immunohematology.
      • GANP, an orthologue of yeast Sac3, prevents R-loop formation and hyper-recombination.
      • Aberrant expression of GANP is frequently observed in various neoplasms.
      • GANP is essential for survival of high-affinity B cells in germinal centers.
      • Augmented GANP leads to Hodgkin lymphomagenesis with B-cell/macrophage biphenotype.
      Germinal center-associated nuclear protein (GANP) is a unique and multifunctional protein that plays a critical role in cell biology, neurodegenerative disorders, immunohematology, and oncogenesis. GANP is an orthologue of Saccharomyces Sac3, one of the components of the transcription export 2 (TREX-2) complex and a messenger RNA (mRNA) nuclear export factor. GANP is widely conserved in all mammals, including humans. Although GANP was originally discovered as a molecule upregulated in the germinal centers of secondary lymphoid follicles in peripheral lymphoid organs, it is expressed ubiquitously in many tissues. It serves numerous functions, including making up part of the mammalian TREX-2 complex; mRNA nuclear export via nuclear pores; prevention of R-loop formation, genomic instability, and hyper-recombination; and B-cell affinity maturation. In this review, we first overview the extensive analyses that have revealed the basic functions of GANP and its ancestor molecule Sac3, including mRNA nuclear export and regulation of R-loop formation. We then describe how aberrant expression of GANP is significantly associated with cancer development. Moreover, we discuss a crucial role for GANP in B-cell development, especially affinity maturation in germinal centers. Finally, we illustrate that overexpression of GANP in B cells leads to lymphomagenesis resembling Hodgkin lymphoma derived from germinal center B cells, and that GANP may be involved in transdifferentiation of B cells to macrophages, which strongly affects Hodgkin lymphomagenesis.
      Germinal center-associated nuclear protein (GANP) is a unique and amazing molecule that has numerous cell biological, neurodegenerative, immunohematological, and oncogenic roles, and is critical for human health.
      We discovered GANP in 2000 as a molecule associated with mouse B-cell differentiation and affinity maturation in germinal centers into secondary lymphoid follicles; in this context, somatic hypermutation of variable-region genes and class switching of B-cell receptors (BCRs) occur in B cells driven against T cell-dependent antigens [
      • Kuwahara K
      • Yoshida M
      • Kondo E
      • et al.
      A novel nuclear phosphoprotein, GANP, is up-regulated in centrocytes of the germinal center and associated with MCM3, a protein essential for DNA replication.
      ,
      • Abe E
      • Kuwahara K
      • Yoshida M
      • et al.
      Structure, expression, and chromosomal localization of the human gene encoding a germinal center-associated nuclear protein (GANP) that associates with MCM3 involved in the initiation of DNA replication.
      ]. We immunohistochemically screened monoclonal antibodies that recognized factors expressed on germinal center B cells and found that one antibody, designated 29–15, reacted to a molecule that was upregulated in germinal centers. Using a λgt11 expression cloning method, we finally identified a novel mouse gene, named ganp. Mouse GANP is a 210-kDa nuclear protein composed of 1,971 amino acids (aa), and the middle portion of mouse GANP bears sequence similarity to Saccharomyces cerevisiae Sac3 (1,301 aa; 23% at amino acid level).
      It is now well known that orthologues of yeast Sac3 are widely conserved in all mammals, including Homo sapiens, where it has come to be known as GANP (Figure 1). Human ganp is located on a chromosome 21q22.3 locus and encodes a 1,980-aa protein [
      • Abe E
      • Kuwahara K
      • Yoshida M
      • et al.
      Structure, expression, and chromosomal localization of the human gene encoding a germinal center-associated nuclear protein (GANP) that associates with MCM3 involved in the initiation of DNA replication.
      ]. In addition to a Sac3 conservative domain, mammalian GANP contains two domains that share sequence similarity with two other mammalian proteins. One domain is approximately 150 aa long and is located on the amino (N)-terminal side of the Sac3 conservative domain, which bears similarity to DNA primase p49 [
      • Kuwahara K
      • Tomiyasu S
      • Fujimura S
      • et al.
      Germinal center-associated nuclear protein (GANP) has a phosphorylation-dependent DNA-primase activity that is up-regulated in germinal center regions.
      ]. The other domain is MCM3AP (MCM3-acetylating protein), which resembles the carboxyl (C)-terminal region of human and mouse GANP [
      • Takei Y
      • Tsujimoto G.
      Identification of a novel MCM3-associated protein that facilitates MCM3 nuclear localization.
      ,
      • Takei Y
      • Swietlik M
      • Tanoue A
      • Tsujimoto G
      • Kouzarides T
      • Laskey R
      MCM3AP, a novel acetyltransferase that acetylates replication protein MCM3.
      ]. We suggest that MCM3AP may be a short form of GANP produced by alternative splicing, although another group reported that mcm3ap was transcribed from a unique promoter (see Figure 1) [
      • Wickramasinghe VO
      • McMurtrie PI
      • Marr J
      • et al.
      MCM3AP is transcribed from a promoter within an intron of the overlapping gene for GANP.
      ]. Confusingly, some research groups use the gene name mcm3ap to refer to the full-length form of ganp, rather than the short form. Historically, human MCM3AP has been reported as a novel 80-kDa protein encoded by map80 [
      • Takei Y
      • Tsujimoto G.
      Identification of a novel MCM3-associated protein that facilitates MCM3 nuclear localization.
      ]. Afterward, the gene symbol was altered from map80 to mcm3ap based on its intrinsic activity as an acetylase [
      • Takei Y
      • Swietlik M
      • Tanoue A
      • Tsujimoto G
      • Kouzarides T
      • Laskey R
      MCM3AP, a novel acetyltransferase that acetylates replication protein MCM3.
      ]. Although the gene name mcm3ap firstly registered was annotated in several databases, many researchers, including us, insist that GANP (210-kDa protein) is a long form of MCM3AP (80-kDa protein). Moreover, the roles and functions of the GANP protein are obviously different from those of MCM3AP [
      • Kuwahara K
      • Yoshida M
      • Kondo E
      • et al.
      A novel nuclear phosphoprotein, GANP, is up-regulated in centrocytes of the germinal center and associated with MCM3, a protein essential for DNA replication.
      ,
      • Abe E
      • Kuwahara K
      • Yoshida M
      • et al.
      Structure, expression, and chromosomal localization of the human gene encoding a germinal center-associated nuclear protein (GANP) that associates with MCM3 involved in the initiation of DNA replication.
      ]. To clear up the confusion, ganp is preferable as the name of the gene that encodes GANP.
      Figure 1
      Figure 1Molecular structures of Sac3/GANP family proteins in several species. (A) Human GANP is a 1,980-aa protein that consists of several functional domains, including phenylalanine–glycine (FG) repeats, a DNA primase region, a Sac3 conservative region, and an acetyltransferase region. The C-terminal region of GANP is designated MCM3AP (MCM3-acetylating protein); this short form has been detected only in humans. (B) Structures of GANP orthologues compared among several species. The DNA primase and acetyltransferase region are detected only in higher eukaryotes. The bottom protein with 1,301 aa from Saccharomyces cerevisiae corresponds to Sac3.
      Over the last 20 years, it has also been revealed that GANP is expressed ubiquitously in the mammalian body not only to facilitate B-cell affinity maturation of lymphocytes in the context of humoral immunity, but also to serve many functions in many tissues. In this review, we focus on the biological significance of GANP, which is especially important for messenger RNA (mRNA) nuclear export, immunohematology of B-cell differentiation, and oncogenesis of hematolymphoid cells.

      Functions of GANP and its related molecules

      It is widely accepted that GANP plays numerous roles, including those that we have already discovered and those that we predict, with impacts in cell biology, neurology, immunohematology, and oncology. Here we summarize GANP's many functions as clearly and comprehensively as possible. In particular, the important hematologic functions of GANP, such as B-cell affinity maturation and B-cell transdifferentiation into macrophages, are discussed later.

      Composition of the TREX-2 complex

      In 1988, Novick et al. [
      • Novick P
      • Osmond BC
      • Botstein D
      Suppressors of yeast actin mutations.
      ] discovered Sac3 (suppressor of actin) in S. cerevisiae, and found that mutant Sac3 decreased the temperature sensitivity of actin gene expression in the act1-1 mutant of this yeast. However, subsequent studies reported that sac3-deficient, act1-1 mutant S. cerevisiae did not exhibit decreased temperature sensitivity of actin gene expression, suggesting that reduction of temperature sensitivity is not simply caused by loss-of-function Sac3 [
      • Bauer A
      • Kölling R.
      Characterization of the SAC3 gene of Sccharomyces cerevisiae.
      ]. At that point, the molecular functions of Sac3 were unknown, although it had been revealed that Sac3-deficient yeast exhibited a cell cycle mitotic delay and chromosome instability [
      • Bauer A
      • Kölling R.
      The SAC3 gene encodes a nuclear protein required for normal progression of mitosis.
      ].
      The turning point came in 2002 when Fischer et al. [
      • Fischer T
      • Strässer K
      • Rácz A
      • et al.
      The mRNA export machinery requires the novel Sac3p-Thp1p complex to dock at the nucleoplasmic entrance of the nuclear pores.
      ] found that Sac3 forms a stable complex with Thp1 and functions in transcription elongation. Interestingly, Sac3 or Thp1 mutation causes a severe defect in mRNA nuclear export. In addition, Sus1, a component of the SAGA complex with histone acetylase activity; Cdc31, a yeast centrin; and Sem1, a component of the ubiquitin/proteasome system, were similarly indispensable for mRNA nuclear export [
      • Rodríguez-Navarro S
      • Fischer T
      • Luo MJ
      • et al.
      Sus1, a functional component of the SAGA histone acetylase complex and the nuclear pore-associated mRNA export machinery.
      ,
      • Fischer T
      • Rodríguez-Navarro S
      • Pereira G
      • Rácz A
      • Schiebel E
      • Hurt E
      Yeast centrin Cdc31 is linked to the nuclear mRNA export machinery.
      ,
      • Faza MB
      • Kemmler S
      • Jimeno S
      • et al.
      Sem1 is a functional component of the nuclear pore complex-associated messenger RNA export machinery.
      ]. Together with these results, it was eventually revealed that Thp1, Sus1, Cdc31, and Sem1 bind to a Sac3 scaffold and form a complex molecule involved in mRNA nuclear export and transcription-coupled DNA recombination. They named the complex of Sac3, Thp1, Sus1, Cdc31, and Sem1 the transcription-export 2 (TREX-2) complex. We should note that 3′ repair exonuclease 2 (TREX2) is a completely different single small protein, and indeed, we have seen descriptions in some papers that “TREX-2 complex” stands for “3′ repair exonuclease-2 complex”; however, this is incorrect.
      It is now understood, as described above, that the protein sequence of Saccharomyces Sac3 is homologous to that of the middle portion of GANP and is evolutionally conserved in sequence and function among several species. In addition, the mammalian TREX-2 complex is thought to have a similar function in mRNA nuclear export. The mammalian TREX-2 complex is composed of GANP, PCID2, ENY2, centrin3/4, and DSS1 (the corresponding orthologues of Saccharomyces spp. are Sac3, Thp1, Sus1, Cdc31, and Sem1, respectively).

      GANP-dependent mRNA nuclear export via nuclear pores

      The TREX-2 complex stably co-localizes with nuclear pore complexes at the nuclear periphery and plays an important role in mRNA nuclear export through the nuclear pore by interacting with NXF1 [
      • Wickramasinghe VO
      • McMurtrie PI
      • Mills AD
      • et al.
      mRNA export from mammalian cell nuclei is dependent on GANP.
      ]. NXF1 (mammalian orthologue of Saccharomyces Mex67) is a nuclear RNA export factor and includes two important domains: one is an RNA-binding domain (RNA recognition motif and leucine-rich repeats) that facilitates association of NXF1 with mRNA to form a messenger ribonucleoprotein (mRNP), and the other is an ubiquitin-associated–like domain that mediates interactions with phenylalanine–glycine (FG) repeat motifs. Because the N-terminal side of GANP contains FG repeat motifs, nuclear pore-localized TREX-2 complex can associate with NXF1-binding mRNPs. Thus, many researchers now accept that GANP is an integral component of the mammalian mRNA nuclear export machinery and facilitates transfer of NXF1-binding mRNPs to nuclear pore complexes [
      • Umlauf D
      • Bonnet J
      • Waharte F
      • et al.
      The human TREX-2 complex is stably associated with the nuclear pore basket.
      ].
      In mammals, however, researchers also believe there exist both GANP-dependent and GANP-independent pathways for transfer of NXF1-binding mRNPs to nuclear pore complexes, and we were the first to recognize this fact. We found that ganp RNA interference (RNAi) in HeLa cells led to cell cycle arrest at the G2/M phase, increased abnormal chromosome alignment of metaphase chromosomes, and finally led to cell apoptosis. These defects followed destabilization of cohesin, a centromeric protein complex that mediates sister chromatid cohesion, caused by a defect in mRNA nuclear export and erroneous translation of shugoshin-1, a molecule that protects centromeric cohesion [
      • Okamoto N
      • Kuwahara K
      • Ohta K
      • et al.
      Germinal center-associated nuclear protein (GANP) is involved in mRNA export of Shugoshin-1 required for centromere cohesion and in sister-chromatid exchange.
      ]. On the other hand, we found that RNAi of not ganp but pcid2 also resulted in a cell cycle abnormality, increased apoptosis, and polyploidy, which were caused by reduced translation of Mad2, a spindle checkpoint protein [
      • Nakaya T
      • Kuwahara K
      • Ohta K
      • et al.
      Critical role of Pcid2 in B cell survival through the regulation of MAD2 expression.
      ]. Based on these results and those from other studies, all transcripts can be separated into GANP/NFX1-dependent and only NFX1-dependent ones. The former may include specific classes of mRNAs, such as RNA synthesis and processing factors that may enable rapid changes in gene expression [
      • Wickramasinghe VO
      • Andrews R
      • Ellis P
      • et al.
      Selective nuclear export of specific classes of mRNA from mammalian nuclei is promoted by GANP.
      ].

      Prevention of R-loop formation, genomic instability, and hyper-recombination

      From 2001 to 2003, Aguilera and colleagues [
      • Gallardo M
      • Aguilera A.
      A new hyperrecombination mutation identifies a novel yeast gene, THP1, connecting transcription elongation with mitotic recombination.
      ,
      • Gallardo M
      • Luna R
      • Erdjument-Bromage H
      • Tempst P
      • Aguilera A
      Nab2p and the Thp1p-Sac3p complex functionally interact at the interface between transcription and mRNA metabolism.
      ,
      • Huertas P
      • Aguilera A.
      Cotranscriptionally formed DNA:RNA hybrids mediate transcription elongation impairment and transcription-associated recombination.
      ] made revolutionary breakthroughs in S. cerevisiae. Not only did they announce that S. cerevisiae deficient in Sac3 or Thp1 exhibit a hyper-recombination phenotype, but they unraveled the mysterious relationship between transcription and recombination during mRNA metabolism by invoking a DNA:RNA hybrid model [
      • Gallardo M
      • Aguilera A.
      A new hyperrecombination mutation identifies a novel yeast gene, THP1, connecting transcription elongation with mitotic recombination.
      ,
      • Gallardo M
      • Luna R
      • Erdjument-Bromage H
      • Tempst P
      • Aguilera A
      Nab2p and the Thp1p-Sac3p complex functionally interact at the interface between transcription and mRNA metabolism.
      ,
      • Huertas P
      • Aguilera A.
      Cotranscriptionally formed DNA:RNA hybrids mediate transcription elongation impairment and transcription-associated recombination.
      ]. When mRNA metabolism, especially mRNA nuclear export, is disturbed, mRNA that cannot move out of the nucleus forms a hybrid with one of two strands of DNA and disturbs the other DNA strand. The DNA:RNA hybrid and the dispersed single DNA strand form an “R-loop” structure that easily results in DNA breaks, genomic instability, and hyper-recombination. The DNA breaks that are induced by R-loop formation, which result from inhibited mRNA nuclear export, are called “transcription-coupled DNA damage.”
      We now know that GANP prevents hyper-recombination in mammals. To establish this fact at the time, we created immortalized mouse embryonic fibroblasts (MEFs) from ganp-heterodeficient mice (ganp+/d MEFs). ganp+/d MEFs exhibited a marked increase in DNA recombination compared with wild-type MEFs, whereas spontaneous DNA recombination was overwhelmingly inhibited by forced overexpression of GANP in NIH3T3 cells. We additionally showed in another experiment that GANP also suppressed DNA recombination in aid-induced NIH3T3 cells, in which activation-induced cytidine deaminase (AID) frequently introduced DNA mutations by changing a cytosine:guanine (C:G) base pair into a uracil:guanine (U:G) mismatch. In summary, the Sac3 conservative domain in GANP can suppress hyper-recombination and contribute to genomic stability in mammalian cells [
      • Yoshida M
      • Kuwahara K
      • Shimasaki T
      • Nakagata N
      • Matsuoka M
      • Sakaguchi N
      GANP suppresses DNA recombination, measured by direct-repeat beta-galactosidase gene construct, but does not suppress the type of recombination applying to immunoglobulin genes in mammalian cells.
      ].

      Prevention of cancerization

      Although little is known about the relationship between oncogenesis and GANP, one of the reasons for oncogenesis may be impairment of mRNA nuclear export machinery caused by ganp dysfunction. Because some mRNA nuclear export is dependent on GANP, as described above, ganp dysfunction may dysregulate some pivotal proteins, such as shugoshin-1, which may affect oncogenesis in various cases. Oncogenesis is also caused by R-loop formations, which induce hyper-recombination and genomic instability.
      It has recently been reported that mRNAs are transported from the nucleus to the cytoplasm through two pathways: a bulk export pathway involving NXF1 (described above in detail) and a specialized pathway involving chromosome region maintenance 1 (CRM1) [
      • Delaleau M
      • Borden KL.
      Multiple export mechanisms for mRNAs.
      ]. Although it is still controversial whether dysregulation of the mRNA nuclear export machinery directly drives cancer initiation, it is well known that a defect in the specialized mRNA export pathway is frequently observed in various cancers [
      • Culjkovic-Kraljacic B
      • Borden KL
      Aiding and abetting cancer: mRNA export and the nuclear pore.
      ]. CRM1 is reportedly overexpressed in a variety of human neoplasms, such as endocervical carcinomas of the uterus and gliomas of the central nervous system, and its overexpression is closely correlated with patient prognosis [
      • van der Watt PJ
      • Maske CP
      • Hendricks DT
      • et al.
      The Karyopherin proteins, Crm1 and Karyopherin beta1, are overexpressed in cervical cancer and are critical for cancer cell survival and proliferation.
      ,
      • Shen A
      • Wang Y
      • Zhao Y
      • Zou L
      • Sun L
      • Cheng C
      Expression of CRM1 in human gliomas and its significance in p27 expression and clinical prognosis.
      ]. Abnormal expression of nucleoporins (Nups) is also detected in cancers; for example, a t(6;9)(p23;q34.1); deknup214 chromosomal translocation is observed in a subtype of acute myeloid leukemia [
      • von Lindern M
      • Fornerod M
      • van Baal S
      • et al.
      The translocation (6;9), associated with a specific subtype of acute myeloid leukemia, results in the fusion of two genes, dek and can, and the expression of a chimeric, leukemia-specific dek-can mRNA.
      ].
      Recently it has been revealed that dysfunction of the bulk mRNA export pathway is also linked to cancer. As described above, the TREX-2 complex facilitates transfer of NXF1-binding mRNPs to nuclear pore complexes and plays a critical role in the bulk mRNA export pathway. GANP, a component of the TREX-2 complex, is frequently over- or underexpressed in various tumors. Overexpression of GANP in tumor cells was observed in lymphomas (see below for details), malignant melanomas, and liver fluke-associated cholangiocarcinomas [
      • Fujimura S
      • Xing Y
      • Takeya M
      • et al.
      Increased expression of germinal center-associated nuclear protein RNA-primase is associated with lymphomagenesis.
      ,
      • Kageshita T
      • Kuwahara K
      • Oka M
      • Ma D
      • Ono T
      • Sakaguchi N
      Increased expression of germinal center-associated nuclear protein (GANP) is associated with malignant transformation of melanocytes.
      ,
      • Chan-On W
      • Kuwahara K
      • Kobayashi N
      • et al.
      Cholangiocarcinomas associated with long-term inflammation express the activation-induced cytidine deaminase and germinal center-associated nuclear protein involved in immunoglobulin V-region diversification.
      ], whereas underexpression was observed in breast cancers and glioblastomas [
      • Kuwahara K
      • Yamamoto-Ibusuki M
      • Zhang Z
      • et al.
      GANP protein encoded on human chromosome 21/mouse chromosome 10 is associated with resistance to mammary tumor development.
      ,
      • Ohta K
      • Kuwahara K
      • Zhang Z
      • et al.
      Decreased expression of germinal center-associated nuclear protein is involved in chromosomal instability in malignant gliomas.
      ]. In the case of breast cancers, we revealed that GANP deficiency is strongly associated with mammary carcinogenesis. Breast cancers frequently occur in two ganp mutant mouse lines, female ganp-heterodeficient mice and mammary-specific ganp-deficient mice. The latter were ganp floxed (fl) (wapcreganpfl/fl) mice, in which a part of ganp was eliminated between two loxP sequences inserted in the 5′-flanking region of exon 2 and the 3′-flanking region of exon 3 using cre/loxP recombination technology. With this method, ganp excision occurred when the wap promotor was activated on the 18th day of pregnancy, cre was subsequently translated, and the loxP DNA sequences were cleaved. After the 18th day of pregnancy, the mammary glands in these mice gradually manifested structural atypia and finally became cancerous. Together with in vitro experiments, these results indicate that GANP can suppress the DNA damage induced by estrogen exposure and has an anti-oncogenic effect on breast carcinogenesis [
      • Kuwahara K
      • Yamamoto-Ibusuki M
      • Zhang Z
      • et al.
      GANP protein encoded on human chromosome 21/mouse chromosome 10 is associated with resistance to mammary tumor development.
      ].

      Nuclear import of MCM3 and regulation of cell cycle

      As described above, the C-terminal region of human and mouse GANP is identical to MCM3AP (MCM3-acetylating protein). MCM3AP is a MCM3-binding molecule that acetylates MCM3 to suppress cell cycle progression and facilitates nuclear import of MCM3 [
      • Takei Y
      • Tsujimoto G.
      Identification of a novel MCM3-associated protein that facilitates MCM3 nuclear localization.
      ,
      • Takei Y
      • Swietlik M
      • Tanoue A
      • Tsujimoto G
      • Kouzarides T
      • Laskey R
      MCM3AP, a novel acetyltransferase that acetylates replication protein MCM3.
      ].

      Pathogenesis of Charcot–Marie–Tooth disease

      It has been reported that mutations in ganp were detected in nine patients and five unrelated families with Charcot–Marie–Tooth disease [
      • Ylikallio E
      • Woldegebriel R
      • Tumiati M
      • et al.
      MCM3AP in recessive Charcot–Marie–Tooth neuropathy and mild intellectual disability.
      ]. Charcot–Marie–Tooth disease is a hereditary neuropathy of the peripheral nervous system characterized by progressive loss of muscle and touch sensation. More than 40 genes have been reported as responsible for Charcot–Marie–Tooth disease, and in 2017, it was discovered that a ganp mutation also causes a variant of this disease [
      • Ylikallio E
      • Woldegebriel R
      • Tumiati M
      • et al.
      MCM3AP in recessive Charcot–Marie–Tooth neuropathy and mild intellectual disability.
      ]. The variant of the disease presents in children, who develop axonal or demyelinating neuropathy; these patients may carry either heterozygous or homozygous mutant ganp alleles. However, according to research using patient fibroblasts, this mutant form of GANP localized normally to the nuclear envelope, and severe GANP dysfunction did not affect DNA repair in vitro, even in patients with a nonsense mutation in ganp. Thus, a role for GANP in the patho-etiology of Charcot–Marie–Tooth disease is still unclear.

      DNA primase activity resembling p49

      As described above, human GANP and mouse GANP contain a 150-aa-long domain on the N-terminal side of the Sac3 conservative domain that is slightly homologous in protein sequence to the DNA primase p49 [
      • Kuwahara K
      • Tomiyasu S
      • Fujimura S
      • et al.
      Germinal center-associated nuclear protein (GANP) has a phosphorylation-dependent DNA-primase activity that is up-regulated in germinal center regions.
      ]. In vitro analysis has revealed that recombinant GANP protein synthesizes RNA primers, resulting in DNA replication in the presence of DNA polymerase I and single-stranded DNA templates [
      • Kuwahara K
      • Tomiyasu S
      • Fujimura S
      • et al.
      Germinal center-associated nuclear protein (GANP) has a phosphorylation-dependent DNA-primase activity that is up-regulated in germinal center regions.
      ]. The DNA primase activity of GANP is controlled by phosphorylation at Ser502 (putative phosphorylation site by Cdk2) and induced by CD40-mediated signaling in vitro or by antigen stimulation in vivo [
      • Kuwahara K
      • Tomiyasu S
      • Fujimura S
      • et al.
      Germinal center-associated nuclear protein (GANP) has a phosphorylation-dependent DNA-primase activity that is up-regulated in germinal center regions.
      ]. It has recently been revealed that Ser502-phosphorylated GANP is preferentially detected in germinal center B cells. In addition to the conventional DNA polymerase α-primase complex, DNA primase in GANP may be essential for excess DNA synthesis to promote somatic hypermutation and affinity maturation in germinal center B cells.

      Immunohematological relationship between GANP and B-cell affinity maturation

      Affinity maturation is now thought to be composed of two steps, somatic hypermutation and clonal selection, which occur in the germinal centers of secondary lymphoid follicles [
      • Bannard O
      • Cyster JG
      Germinal centers: programmed for affinity maturation and antibody diversification.
      ]. Somatic hypermutation is a programmed process for introducing mutations and affects IgV, a part of the antigen-binding coding sequences of Ig genes. The mutations promote the diversity of antibodies by altering their binding specificity and affinities. Clonal selection is the process by which B cells that react with no antigens or some antigens only very weakly are eliminated; these defective B cells are selected and made to undergo apoptosis by follicular helper T cells in the germinal centers. It is now known that GANP is an essential immunohematological molecule that maintains immune function by playing an important role in somatic hypermutation and probably in clonal selection of antigen-specific B cells that express high-affinity BCRs.

      Germinal center formation and somatic hypermutation

      The functions of GANP during germinal center formation and affinity maturation have been clarified using laboratory mouse models [
      • Kuwahara K
      • Fujimura S
      • Takahashi Y
      • et al.
      Germinal center-associated nuclear protein contributes to affinity maturation of B cell antigen receptor in T cell-dependent responses.
      ]. Unfortunately, however, ganp-homodeficient mice manifested embryonic lethality by around embryonic day 11.5, presumably caused by abnormal morphogenesis of the brain and heart (unpublished data). In addition, it was difficult to establish a hematopoietic system–specific ganp-deficient mouse that was also engineered to reconstitute ganp-deficient fetal liver cells into immunocompromised mice, as very few liver cells could be obtained from homozygous embryos.
      Hence, we established B cell–specific ganp-deficient (cd19creganpfl/fl) mice using cre/loxP recombination technology [
      • Kuwahara K
      • Fujimura S
      • Takahashi Y
      • et al.
      Germinal center-associated nuclear protein contributes to affinity maturation of B cell antigen receptor in T cell-dependent responses.
      ]. Compared with wild-type mice, cd19-cre-ganpfl/fl mice exhibited normal B-cell number, development, and subpopulations; serum Ig levels;mitogen-induced B-cell proliferation in vitro; immune responses against T cell–independent antigen; and B-cell class switching, whereas germinal center formation was retarded even on immunization with T cell–dependent antigen. Moreover, somatic mutations in IgV and high-affinity variant induction (33Trp→33Leu) of the VH186.2 region occurred less frequently in cd19creganpfl/fl mice than that in wild-type mice as a response to immune reaction of a hapten of nitrophenyl-chicken γ-globulin [
      • Kuwahara K
      • Fujimura S
      • Takahashi Y
      • et al.
      Germinal center-associated nuclear protein contributes to affinity maturation of B cell antigen receptor in T cell-dependent responses.
      ].
      We also established and analyzed ganp transgenic C57BL/6 (Ig-ganpTg) mice, in which GANP was overexpressed in B cells [
      • Sakaguchi N
      • Kimura T
      • Matsushita S
      • et al.
      Generation of high-affinity antibody against T cell-dependent antigen in the ganp gene-transgenic mouse.
      ]. Ig-ganpTg mice had a normal phenotype in terms of B-cell number, development, and subpopulations; serum Ig levels; and immune responses against T cell–independent antigens; however, antibodies against a hapten of nitrophenyl exhibited much higher affinity in Ig-ganpTg mice than they did in wild-type C57BL/6 mice. Ig-ganpTg mice were also able to produce extremely high-affinity anti-human immunodeficiency virus-1 monoclonal antibodies with neutralizing activity. These results clearly indicated that GANP is essential for production or maintenance of high-affinity B cells in germinal centers.
      It has been reported that GANP may be essential for transporting AID to B-cell nuclei for targeting IgV [
      • Maeda K
      • Singh SK
      • Eda K
      • et al.
      GANP-mediated recruitment of activation-induced cytidine deaminase to cell nuclei and to immunoglobulin variable region DNA.
      ]. However, whether GANP deficiency leads to impairment of somatic hypermutation remains controversial.

      Double-strand breaks at immunoglobulin variable regions

      Furthermore, using ligation-mediated polymerase chain reaction, we found that double-strand breaks at IgV were more frequently observed in Ig-ganpTg B cells and less frequently observed in ganp-deficient B cells, compared with control B cells [
      • Kawatani Y
      • Igarashi H
      • Matsui T
      • et al.
      Cutting edge: double-stranded DNA breaks in the IgV region gene were detected at lower frequency in affinity-maturation impeded GANP–/– mice.
      ]. These results suggested that the number of IgV double-strand breaks in germinal center B cells is positively correlated with GANP expression. Double-strand breaks at IgV induced by GANP may be required for affinity maturation, and lack of GANP may cause impaired affinity maturation of antigen-driven B cells and subsequently trigger B-cell apoptosis as clonal selection controlled by follicular dendritic cells. Note that GANP induces double-strand breaks only at IgV and, opposingly, avoids transcription-coupled DNA damage in the other genome. It is speculated that double-strand DNA breaks at IgV are phenomenologically unrelated to mRNA nuclear export, R-loop formation, and DNA primase activity; however, this idea remains controversial.

      Modulation of GANP expression by the upstream molecule Lyn

      GANP is a molecule located downstream of Lyn, an src-type tyrosine kinase [
      • Hibbs ML
      • Tarlinton DM
      • Armes J
      • et al.
      Multiple defects in the immune system of Lyn-deficient mice, culminating in autoimmune disease.
      ]. Lyn plays a role in signal transduction of intracytoplasmic molecules (such as surface IgM [sIgM] and CD40) in B cells in the peripheral lymphoid organs [
      • Hibbs ML
      • Tarlinton DM
      • Armes J
      • et al.
      Multiple defects in the immune system of Lyn-deficient mice, culminating in autoimmune disease.
      ]. Lyn-deficient (lyn−/−) mice have impaired development of germinal centers in the spleen and decreased antibody affinity, and GANP expression is decreased in Lyn-deficient chicken DT40 cells and mouse B cells [
      • Mirnics ZK
      • Caudell E
      • Gao Y
      • et al.
      Microarray analysis of Lyn-deficient B cells reveals germinal center-associated nuclear protein and other genes associated with the lymphoid germinal center.
      ]. These results suggest that Lyn may control formation and proliferation of germinal centers via GANP. Moreover, both Ig-ganpTglyn−/− mice and lyn−/− mice exhibited similar B-cell differentiation, serum Ig levels, and impaired germinal center formation phenotypes, although mature B cells, similar to germinal center B cells, were partly rescued and affinity maturation was potentially recovered in Ig-ganpTglyn−/− mice [
      • Kuwahara K
      • Nakaya T
      • Phimsen S
      • et al.
      Lyn signaling to upregulate GANP is critical for the survival of high-affinity B cells in germinal centers of lymphoid organs.
      ]. These results indicate that GANP is indispensable for germinal center formation, but plays a critical role in Lyn-mediated signaling for the clonal selection of B cells in peripheral lymphoid organs (Figure 2A).
      Figure 2
      Figure 2Schematic diagrams of the role of GANP during B-cell maturation and differentiation. (A) Antigen stimulation induces tyrosine phosphorylation of src-family kinases, including Lyn, followed by activation of downstream molecules. The mouse ganp promoter region contains a PU.1-binding site, and GANP expression is regulated by Lyn through PU.1. The introduction of the ganp gene into lyn−/− mice did not restore germinal center formation; however, the number of germinal center-like B cells with high-affinity antibodies was partly rescued out of the germinal center. (B) Antigen-stimulated B cells rapidly proliferate in germinal centers and become centroblasts, in which B-cell receptor is downregulated. Afterwards, centroblasts differentiate into centrocytes, which are subject to affinity maturation with help from follicular dendritic cells and follicular helper T cells. Hodgkinoid lymphoma cells developed in Ig-ganpTg mice exhibit a B-cell/macrophage biphenotype, an intermediate state between B cell and macrophage.

      Hodgkin lymphomagenesis and transdifferentiation between B cells and macrophages

      Transdifferentiation, also known as reprogramming, is the process by which a differentiated somatic cell transforms into another type of differentiated cell without undergoing an intermediate pluripotent state or dedifferentiating into a progenitor cell.
      Hodgkin lymphoma is a lymphoid neoplasm characterized by the development of large dysplastic lymphocytes called Hodgkin and Reed–Sternberg cells, which originate from B cells located in the germinal centers of peripheral lymphoid organs and may or may not be associated with Epstein–Barr virus infection. Interestingly, Hodgkin lymphoma cells exhibit altered characteristics, such as partial loss of B-cell lineage markers, including surface IgM (sIgM), CD20, and CD79a, and sometimes express biphenotypic characteristics of B cells and macrophages, such as phagocytic activity and macrophage-derived cytokine secretion [
      • Sakai Y
      • Rezano A
      • Okada S
      • et al.
      The novel cytological model of B-cell/macrophage biphenotypic cell Hodgkin lymphoma in ganp-transgenic mice.
      ].
      Our research using lyn−/− and Ig-ganpTg mice indicated that GANP regulates cell transdifferentiation between B cells and macrophages [
      • Sakai Y
      • Rezano A
      • Okada S
      • et al.
      The novel cytological model of B-cell/macrophage biphenotypic cell Hodgkin lymphoma in ganp-transgenic mice.
      ]. Moreover, GANP overexpression may invoke Hodgkin lymphoma, which exhibits a B-cell/macrophage biphenotype [
      • Sakai Y
      • Rezano A
      • Okada S
      • et al.
      The novel cytological model of B-cell/macrophage biphenotypic cell Hodgkin lymphoma in ganp-transgenic mice.
      ].

      Transdifferentiational B-cell/macrophage biphenotypic cells in lyn−/−mice

      B-Cell/macrophage biphenotypic cells, which express both Ig and macrophage-specific marker CD11b, appear in increased numbers in the spleens of lyn−/− mice compared with those in lyn+/− mice [
      • Sakaguchi N
      • Kimura T
      • Matsushita S
      • et al.
      Generation of high-affinity antibody against T cell-dependent antigen in the ganp gene-transgenic mouse.
      ,
      • Sakai Y
      • Rezano A
      • Okada S
      • et al.
      The novel cytological model of B-cell/macrophage biphenotypic cell Hodgkin lymphoma in ganp-transgenic mice.
      ]. Approximately one-third of CD11b+ cells in the spleens of 14-week-old lyn−/− mice were cytoplasmic IgM (cIgM) positive, although cIgM+CD11b+ cells were still observed less frequently in 8-week-old lyn−/− mice [
      • Sakai Y
      • Rezano A
      • Okada S
      • et al.
      The novel cytological model of B-cell/macrophage biphenotypic cell Hodgkin lymphoma in ganp-transgenic mice.
      ]. Furthermore, in Ig-ganpTglyn−/− mice, the cell population of cIgM+CD11b+ cells in the spleen was almost normal [
      • Sakai Y
      • Rezano A
      • Okada S
      • et al.
      The novel cytological model of B-cell/macrophage biphenotypic cell Hodgkin lymphoma in ganp-transgenic mice.
      ]. Thus, biphenotypic cIgM+CD11b+ cells were mostly observed in lyn−/− mice but not in control or Ig-ganpTglyn−/− mice, which indicates that the biphenotypic cells may be B cells incompletely transdifferentiated to macrophages by lack of GANP and that GANP regulates cell transdifferentiation from B cells to macrophages in a Lyn-independent manner.

      Hodgkinoid lymphomas in Ig-ganpTg mice

      B-Cell lymphomas frequently develop in the livers and spleens of Ig-ganpTg mice after prolonged observation [
      • Sakai Y
      • Rezano A
      • Okada S
      • et al.
      The novel cytological model of B-cell/macrophage biphenotypic cell Hodgkin lymphoma in ganp-transgenic mice.
      ]. They exhibited abnormally large and irregularly shaped B cells. Astonishingly, these cells exhibited B-cell/macrophage biphenotypic hodgkinoid characteristics that resemble human Hodgkin lymphoma. In addition, although biphenotypic hodgkinoid cells were originated from B cells expressing rearranged μ-heavy/κ-light chains and cytoplasmic CD45R (B220), they not only eliminated sIgM and surface CD45R(B220) but also acquired macrophage-specific characteristics, such as major histocompatibility complex class II, F4/80, CD68, and CD204 [
      • Fujimura S
      • Xing Y
      • Takeya M
      • et al.
      Increased expression of germinal center-associated nuclear protein RNA-primase is associated with lymphomagenesis.
      ]. Moreover, the hodgkinoid cell line established from Ig-ganpTg mice exhibited high phagocytotic activity in vitro and secreted both cytokines (granulocyte/macrophage colony-stimulating factor [GM-CSF], macrophage colony-stimulating factor [M-CSF], interleukin [IL]-4, IL-10, IL-12, IL-13, tumor necrosis factor [TNF]-α, vascular endothelial growth factor, and thrombopoietin) and CC chemokines (monocyte chemoattractant protein [MCP]-1, MCP-5, keratinocyte chemoattractant, and RANTES) [
      • Sakai Y
      • Rezano A
      • Okada S
      • et al.
      The novel cytological model of B-cell/macrophage biphenotypic cell Hodgkin lymphoma in ganp-transgenic mice.
      ]. Collectively, these findings suggest that these hodgkinoid cells exhibit the functions of macrophages and that GANP may be associated with Hodgkin lymphomagenesis and transdifferentiation from B cells to macrophages.
      The Ig-ganpTg mouse, a novel cytological model of Hodgkin lymphoma, is quietly beneficial. Approximately half of human Hodgkin lymphoma cases are caused by Epstein–Barr virus (EBV) infection; however, EBV cannot infect mice and immortalize B cells[
      • Küppers R
      • Schwering I
      • Bräuninger A
      • Rajewsky K
      • Hansmann ML
      Biology of Hodgkin's lymphoma.
      ]. GANP is known to operate downstream of CD40, and CD40 activates NF-κB signaling constitutively in Hodgkin and Reed–Sternberg cells. Ig-ganpTg mice are the only mice in which this signaling cascade may be critical for Hodgkin lymphomagenesis. Further studies using Ig-ganpTg mice are required to fully elucidate the mechanisms of Hodgkin lymphomagenesis.

      Hodgkin lymphomas in humans

      GANP is overexpressed in human Hodgkin lymphoma cells, as well as Hodgkinoid lymphoma cells developed in Ig-ganpTg mice [
      • Sakai Y
      • Rezano A
      • Okada S
      • et al.
      The novel cytological model of B-cell/macrophage biphenotypic cell Hodgkin lymphoma in ganp-transgenic mice.
      ]. Because GANP overexpression is closely related to oncogenesis of Hodgkin lymphoma, which is derived from germinal center B cells, GANP plays a critical role not only in affinity maturation of B cells but also in transdifferentiation from B cells to macrophages in the germinal centers, even in humans (Figure 2B). In addition, the upstream or downstream molecules of GANP may conceivably be alternative therapeutic targets of human Hodgkin lymphoma.

      Conclusion

      GANP has a structure homologous to that of yeast Sac3 and is a member of the TREX-2 complex. It has been documented as a critical regulator of transcription-coupled DNA damage associated with mRNA metabolism. We originally identified GANP as a molecule that is upregulated in the germinal centers of peripheral lymphoid organs and have focused on its functional role in the immunohematological field using in vivo studies of mutant mice for two decades. GANP functions can be partly explained by transcription-coupled DNA damage; however, its complex in vivo functions are still not fully understood, as the ganp gene contains many functional domains and exhibits a complicated array of interactions with other molecules. As a consequence, it may be difficult to further study GANP like other macromolecules.
      In this review, we also introduced a role for GANP in modulating oncogenesis. Using mouse models, we showed that GANP expression is not simply enhanced in various tumors, but may also be directly or indirectly involved in driving oncogenesis. Why GANP is overexpressed or underexpressed, depending on tumor histological type, remains an important unanswered question. It is crucial that we reveal how GANP differentially affects oncogenesis in hematological malignancies, malignant melanoma, and cholangiocarcinoma in contrast to breast cancers and high-grade gliomas. Further investigations should be conducted to unravel the details of this complex situation.

      Acknowledgments

      We thank Professor Nobuo Sakaguchi for supervising several research projects concerning GANP. We also thank Professor Makoto Kuroda for encouragement. Submission of the review article was supported by a Grant-in-Aid for Young Scientists 20K16228 from the Japan Society for the Promotion of Science (YS). SP was supported by a scholarship from the Japanese Ministry of Education, Culture, Sports, Science and Technology (MEXT).

      Conflict of interest disclosure

      The authors have no conflicts of interest to declare.

      References

        • Kuwahara K
        • Yoshida M
        • Kondo E
        • et al.
        A novel nuclear phosphoprotein, GANP, is up-regulated in centrocytes of the germinal center and associated with MCM3, a protein essential for DNA replication.
        Blood. 2000; 95: 2321-2328
        • Abe E
        • Kuwahara K
        • Yoshida M
        • et al.
        Structure, expression, and chromosomal localization of the human gene encoding a germinal center-associated nuclear protein (GANP) that associates with MCM3 involved in the initiation of DNA replication.
        Gene. 2000; 255: 219-227
        • Kuwahara K
        • Tomiyasu S
        • Fujimura S
        • et al.
        Germinal center-associated nuclear protein (GANP) has a phosphorylation-dependent DNA-primase activity that is up-regulated in germinal center regions.
        Proc Natl Acad Sci USA. 2001; 98: 10279-10283
        • Takei Y
        • Tsujimoto G.
        Identification of a novel MCM3-associated protein that facilitates MCM3 nuclear localization.
        J Biol Chem. 1998; 273: 22177-22180
        • Takei Y
        • Swietlik M
        • Tanoue A
        • Tsujimoto G
        • Kouzarides T
        • Laskey R
        MCM3AP, a novel acetyltransferase that acetylates replication protein MCM3.
        EMBO Rep. 2001; 2: 119-123
        • Wickramasinghe VO
        • McMurtrie PI
        • Marr J
        • et al.
        MCM3AP is transcribed from a promoter within an intron of the overlapping gene for GANP.
        J Mol Biol. 2011; 406: 355-361
        • Novick P
        • Osmond BC
        • Botstein D
        Suppressors of yeast actin mutations.
        Genetics. 1989; 121: 659-674
        • Bauer A
        • Kölling R.
        Characterization of the SAC3 gene of Sccharomyces cerevisiae.
        Yeast. 1996; 12: 965-975
        • Bauer A
        • Kölling R.
        The SAC3 gene encodes a nuclear protein required for normal progression of mitosis.
        J Cell Sci. 1996; 109: 1575-1583
        • Fischer T
        • Strässer K
        • Rácz A
        • et al.
        The mRNA export machinery requires the novel Sac3p-Thp1p complex to dock at the nucleoplasmic entrance of the nuclear pores.
        EMBO J. 2002; 21: 5843-5852
        • Rodríguez-Navarro S
        • Fischer T
        • Luo MJ
        • et al.
        Sus1, a functional component of the SAGA histone acetylase complex and the nuclear pore-associated mRNA export machinery.
        Cell. 2004; 116: 75-86
        • Fischer T
        • Rodríguez-Navarro S
        • Pereira G
        • Rácz A
        • Schiebel E
        • Hurt E
        Yeast centrin Cdc31 is linked to the nuclear mRNA export machinery.
        Nat Cell Biol. 2004; 6: 840-848
        • Faza MB
        • Kemmler S
        • Jimeno S
        • et al.
        Sem1 is a functional component of the nuclear pore complex-associated messenger RNA export machinery.
        J Cell Biol. 2009; 184: 833-846
        • Wickramasinghe VO
        • McMurtrie PI
        • Mills AD
        • et al.
        mRNA export from mammalian cell nuclei is dependent on GANP.
        Curr Biol. 2010; 20: 25-31
        • Umlauf D
        • Bonnet J
        • Waharte F
        • et al.
        The human TREX-2 complex is stably associated with the nuclear pore basket.
        J Cell Sci. 2013; 126: 2656-2667
        • Okamoto N
        • Kuwahara K
        • Ohta K
        • et al.
        Germinal center-associated nuclear protein (GANP) is involved in mRNA export of Shugoshin-1 required for centromere cohesion and in sister-chromatid exchange.
        Genes Cells. 2010; 15: 471-484
        • Nakaya T
        • Kuwahara K
        • Ohta K
        • et al.
        Critical role of Pcid2 in B cell survival through the regulation of MAD2 expression.
        J Immunol. 2010; 185: 5180-5187
        • Wickramasinghe VO
        • Andrews R
        • Ellis P
        • et al.
        Selective nuclear export of specific classes of mRNA from mammalian nuclei is promoted by GANP.
        Nucleic Acids Res. 2014; 42: 5059-5071
        • Gallardo M
        • Aguilera A.
        A new hyperrecombination mutation identifies a novel yeast gene, THP1, connecting transcription elongation with mitotic recombination.
        Genetics. 2001; 157: 79-89
        • Gallardo M
        • Luna R
        • Erdjument-Bromage H
        • Tempst P
        • Aguilera A
        Nab2p and the Thp1p-Sac3p complex functionally interact at the interface between transcription and mRNA metabolism.
        J Biol Chem. 2003; 278: 24225-24232
        • Huertas P
        • Aguilera A.
        Cotranscriptionally formed DNA:RNA hybrids mediate transcription elongation impairment and transcription-associated recombination.
        Mol Cell. 2003; 12: 711-721
        • Yoshida M
        • Kuwahara K
        • Shimasaki T
        • Nakagata N
        • Matsuoka M
        • Sakaguchi N
        GANP suppresses DNA recombination, measured by direct-repeat beta-galactosidase gene construct, but does not suppress the type of recombination applying to immunoglobulin genes in mammalian cells.
        Genes Cells. 2007; 12: 1205-1213
        • Delaleau M
        • Borden KL.
        Multiple export mechanisms for mRNAs.
        Cells. 2015; 4: 452-473
        • Culjkovic-Kraljacic B
        • Borden KL
        Aiding and abetting cancer: mRNA export and the nuclear pore.
        Trends Cell Biol. 2013; 23: 328-335
        • van der Watt PJ
        • Maske CP
        • Hendricks DT
        • et al.
        The Karyopherin proteins, Crm1 and Karyopherin beta1, are overexpressed in cervical cancer and are critical for cancer cell survival and proliferation.
        Int J Cancer. 2009; 124: 1829-1840
        • Shen A
        • Wang Y
        • Zhao Y
        • Zou L
        • Sun L
        • Cheng C
        Expression of CRM1 in human gliomas and its significance in p27 expression and clinical prognosis.
        Neurosurgery. 2009; 65: 153-159
        • von Lindern M
        • Fornerod M
        • van Baal S
        • et al.
        The translocation (6;9), associated with a specific subtype of acute myeloid leukemia, results in the fusion of two genes, dek and can, and the expression of a chimeric, leukemia-specific dek-can mRNA.
        Mol Cell Biol. 1992; 12: 1687-1697
        • Fujimura S
        • Xing Y
        • Takeya M
        • et al.
        Increased expression of germinal center-associated nuclear protein RNA-primase is associated with lymphomagenesis.
        Cancer Res. 2005; 65: 5925-5934
        • Kageshita T
        • Kuwahara K
        • Oka M
        • Ma D
        • Ono T
        • Sakaguchi N
        Increased expression of germinal center-associated nuclear protein (GANP) is associated with malignant transformation of melanocytes.
        J Dermatol Sci. 2006; 42: 55-63
        • Chan-On W
        • Kuwahara K
        • Kobayashi N
        • et al.
        Cholangiocarcinomas associated with long-term inflammation express the activation-induced cytidine deaminase and germinal center-associated nuclear protein involved in immunoglobulin V-region diversification.
        Int J Oncol. 2009; 35: 287-295
        • Kuwahara K
        • Yamamoto-Ibusuki M
        • Zhang Z
        • et al.
        GANP protein encoded on human chromosome 21/mouse chromosome 10 is associated with resistance to mammary tumor development.
        Cancer Sci. 2016; 107: 469-477
        • Ohta K
        • Kuwahara K
        • Zhang Z
        • et al.
        Decreased expression of germinal center-associated nuclear protein is involved in chromosomal instability in malignant gliomas.
        Cancer Sci. 2009; 100: 2069-2076
        • Ylikallio E
        • Woldegebriel R
        • Tumiati M
        • et al.
        MCM3AP in recessive Charcot–Marie–Tooth neuropathy and mild intellectual disability.
        Brain. 2017; 140: 2093-2103
        • Bannard O
        • Cyster JG
        Germinal centers: programmed for affinity maturation and antibody diversification.
        Curr Opin Immunol. 2017; 45: 21-30
        • Kuwahara K
        • Fujimura S
        • Takahashi Y
        • et al.
        Germinal center-associated nuclear protein contributes to affinity maturation of B cell antigen receptor in T cell-dependent responses.
        Proc Natl Acad Sci USA. 2004; 101: 1010-1015
        • Sakaguchi N
        • Kimura T
        • Matsushita S
        • et al.
        Generation of high-affinity antibody against T cell-dependent antigen in the ganp gene-transgenic mouse.
        J Immunol. 2005; 174: 4485-4494
        • Maeda K
        • Singh SK
        • Eda K
        • et al.
        GANP-mediated recruitment of activation-induced cytidine deaminase to cell nuclei and to immunoglobulin variable region DNA.
        J Biol Chem. 2010; 285: 23945-23953
        • Kawatani Y
        • Igarashi H
        • Matsui T
        • et al.
        Cutting edge: double-stranded DNA breaks in the IgV region gene were detected at lower frequency in affinity-maturation impeded GANP–/– mice.
        J Immunol. 2005; 175: 5615-5618
        • Hibbs ML
        • Tarlinton DM
        • Armes J
        • et al.
        Multiple defects in the immune system of Lyn-deficient mice, culminating in autoimmune disease.
        Cell. 1995; 83: 301-311
        • Mirnics ZK
        • Caudell E
        • Gao Y
        • et al.
        Microarray analysis of Lyn-deficient B cells reveals germinal center-associated nuclear protein and other genes associated with the lymphoid germinal center.
        J Immunol. 2004; 172: 4133-4141
        • Kuwahara K
        • Nakaya T
        • Phimsen S
        • et al.
        Lyn signaling to upregulate GANP is critical for the survival of high-affinity B cells in germinal centers of lymphoid organs.
        J Immunol. 2012; 189: 3472-3479
        • Sakai Y
        • Rezano A
        • Okada S
        • et al.
        The novel cytological model of B-cell/macrophage biphenotypic cell Hodgkin lymphoma in ganp-transgenic mice.
        Cancers. 2020; 12: 204
        • Küppers R
        • Schwering I
        • Bräuninger A
        • Rajewsky K
        • Hansmann ML
        Biology of Hodgkin's lymphoma.
        Ann Oncol. 2002; 13: 11-18