Rapid Screening Using Pathomorphologic Interpretation to Detect BRAFV600E Mutation and Microsatellite Instability in Colorectal Cancer.

PURPOSE: Rapid decision-making is essential in precision medicine for initiating molecular targeted therapy for patients with cancer. This study aimed to extract pathomorphologic features that enable the accurate prediction of genetic abnormalities in cancer from hematoxylin and eosin images using deep learning (DL). EXPERIMENTAL DESIGN: A total of 1,657 images (one representative image per patient) of thin formalin-fixed, paraffin-embedded tissue sections from either primary or metastatic tumors with next-generation sequencing-confirmed genetic abnormalities-including BRAFV600E and KRAS mutations, and microsatellite instability high (MSI-H)-that are directly relevant to therapeutic strategies for advanced colorectal cancer were obtained from the nationwide SCRUM-Japan GI-SCREEN project. The images were divided into three groups of 986, 248, and 423 images to create one training and two validation cohorts, respectively. Pathomorphologic feature-prediction DL models were first developed on the basis of pathomorphologic features. Subsequently, gene-prediction DL models were constructed for all possible combinations of pathomorphologic features that enabled the prediction of gene abnormalities based on images filtered by the combination of pathomorphologic feature-prediction models. RESULTS: High accuracies were achieved, with AUCs > 0.90 and 0.80 for 12 and 27, respectively, of 33 analyzed pathomorphologic features, with high AUCs being yielded for both BRAFV600E (0.851 and 0.859) and MSI-H (0.923 and 0.862). CONCLUSIONS: These findings show that novel next-generation pathology methods can predict genetic abnormalities without the need for standard-of-care gene tests, and this novel next-generation pathology method can be applied for colorectal cancer treatment planning in the near future.

Higher plant RecA-like protein is homologous to RadA.

A novel RecA-like protein, differing from Dmc1 and Rad51, was characterized in Oryza sativa L. cv. Nipponbare. Because the protein is homologous to bacterial RadA, the gene was designated OsRadA. The open reading frame was predicted to encode a 66kDa protein of 619 amino acid residues and was found in plants but not animals or yeast. OsRadA showed D-loop and single-stranded DNA-dependent ATPase activities. Gene expression was found to be high in meristematic tissues, and was localized in the nucleus. An RNAi mutant of Arabidopsis thaliana RadA (AtRadA) was sensitive to mutagenic agents such as UV and MMC, suggesting that RadA functions in DNA repair.

Two types of replication protein A in seed plants.

Replication protein A (RPA), a heterotrimeric protein composed of 70, 32 and 14-kDa subunits, has been shown to be essential for DNA replication, repair, recombination, and transcription. Previously, we found that, in two seed plants, rice and Arabidopsis, there are two different types of RPA70-kDa subunit. Substantial biochemical and genetic characterization of these two subunits, termed OsRPA70a and OsRPA70b or AtRPA70a and AtRPA70b, respectively, is described in this report. Inactivation of AtRPA70a by transfer DNA insertion or RNA interference is lethal, so the complex containing RPA70a may be essential for DNA replication. Transfer DNA insertion and RNAi lines for AtRPA70b are morphologically normal, albeit hypersensitive to certain mutagens, such as UV-B and methyl methanesulfonate, suggesting that RPA70b functions mostly in DNA repair. In two-hybrid, pull-down and coexpression analysis, OsRPA70b was found to interact more selectively than OsRPA70a with OsRPA32. The data suggest that two different types of RPA heterotrimer are present in seed plants, and that there may be additional 32 and 14-kDa subunit homologs that interact with OsRPA70a. Each of the two probable plant RPA complexes may have different roles in DNA metabolism.

Characterization of the origin recognition complex (ORC) from a higher plant, rice (Oryza sativa L.).

The origin recognition complex (ORC) protein plays a critical role in DNA replication through binding to sites (origins) where replication commences. The protein is composed of six subunits (ORC1 to 6) in animals and yeasts. Our knowledge of the ORC protein in plants is, however, much less complete. We have performed cDNA cloning and characterization of ORC subunits in rice (Oryza sativa L. cv. Nipponbare) in order to facilitate study of plant DNA replication mechanisms. Our previous report provided a description of a gene, ORC1 (OsORC1), that encodes one of the protein subunits. The present report extends this initial analysis to include the genes that encode four other rice ORC subunits, OsORC2, 3, 4 and 5. Northern hybridization analyses demonstrated the presence of abundant transcripts for all OsORC subunits in shoot apical meristems (SAM) and cultured cells, but not in mature leaves. Interestingly, only OsORC5 showed high levels of expression in organs in which cell proliferation is not active, such as flag leaves, the ears and the non-tip roots. The pattern of expression of OsORC2 also differed from other OsORC subunits. When cell proliferation was temporarily halted for 6-10 days by removal of sucrose from the growth medium, expression of OsORC1, OsORC3, OsORC4 and OsORC5 was substantially reduced. However, the level of expression of OsORC2 remained constant. We suggest from these results that expression of OsORC1, 3, 4 and 5 are correlated with cell proliferation, but the expression of OsORC2 is not.

Interaction between proliferating cell nuclear antigen (PCNA) and a DnaJ induced by DNA damage.

Proliferating cell nuclear antigen (PCNA) is an essential protein for both DNA replication and DNA repair. In the present study using two-hybrid analysis with PCNA from rice, Oryza sativa L. cv. Nipponbare (OsPCNA), we found that OsPCNA interacted with rice DnaJ protein. We have identified DnaJ and designated it as OsDnaJ. OsDnaJ was able to bind to OsPCNA in vitro. Transcripts of OsDnaJ were found to be strongly expressed in the proliferating cells. mRNA of DnaJ was induced by UV and DNA-damaging agents such as H2O2. The expression patterns of OsPCNA were almost the same as OsDnaJ. The relationship between OsPCNA and OsDnaJ is discussed.

A novel interaction between calreticulin and ubiquitin-like nuclear protein in rice.

Calreticulin (CRT), a major Ca2+ -sequestering protein, has been implicated in a variety of cellular functions such as Ca2+ storage, signaling and chaperone activity within the cytoplasm and endoplasmic reticulum. To investigate the biological role of CRT in rice, 21 partial cDNAs, encoding proteins that interacted with rice CRT in a yeast two-hybrid interaction-cloning system, were characterized and the nucleotide sequences were found to be identical to each other. A full-length cDNA of 3.5 kb, obtained from rice genomic sequence data and 5' RACE, codes for a novel protein of 966 amino acid residues and was designated as CRTintP (CRT interacting protein). Primary sequence analysis of CRTintP showed no sequence homology with the known functional proteins; however, a potential ubiquitin-like domain at the N-terminal together with a putative leucine zipper, a nuclear localization signal and several sites for serine/threonine kinases were evident. Cellular localization of CRTintP demonstrated its role in directing green fluorescent protein to the nucleus in onion epidermal cells. Northern and immunoblot analysis showed increased expression of CRT and CRTintP in response to cold stress. Co-immunoprecipitation using anti-CRT antibodies confirmed the existence of the CRT-CRTintP complex in vivo in the stressed leaf tissue, suggesting their potential role in regulating stress response.

DNA repair in higher plants; photoreactivation is the major DNA repair pathway in non-proliferating cells while excision repair (nucleotide excision repair and base excision repair) is active in proliferating cells.

We investigated expression patterns of DNA repair genes such as the CPD photolyase, UV-DDB1, CSB, PCNA, RPA32 and FEN-1 genes by northern hybridization analysis and in situ hybridization using a higher plant, rice (Oryza sativa L. cv. Nipponbare). We found that all the genes tested were expressed in tissues rich in proliferating cells, but only CPD photolyase was expressed in non-proliferating tissue such as the mature leaves and elongation zone of root. The removal of DNA damage, cyclobutane pyrimidine dimers and (6-4) photoproducts, in both mature leaves and the root apical meristem (RAM) was observed after UV irradiation under light. In the dark, DNA damage in mature leaves was not repaired efficiently, but that in the RAM was removed rapidly. Using a rice 22K custom oligo DNA microarray, we compared global gene expression patterns in the shoot apical meristem (SAM) and mature leaves. Most of the excision repair genes were more strongly expressed in SAM. These results suggested that photoreactivation is the major DNA repair pathway for the major UV-induced damage in non-proliferating cells, while both photoreactivation and excision repair are active in proliferating cells.

Characterization of Rad6 from a higher plant, rice (Oryza sativa L.) and its interaction with Sgt1, a subunit of the SCF ubiquitin ligase complex.

We report here the existence of interactions between a ubiquitin-conjugating enzyme, Rad6, from rice, Oryza sativa L. cv. Nipponbare (OsRad6), and Sgt1 (OsSgt1), a novel subunit of the SCF ubiquitin ligase complex. Rad6 is not only related to post-replicational repair but also to the proteasome system, while Sgt1 has a function in kinetochore assembly. The relationship between the two is unexpected, but of great interest. The open reading frames of OsRad6 and OsSgt1 encode predicted products of 152 and 367 amino acid residues, respectively, with molecular weights of 17.3 and 40.9kDa. Two-hybrid and pull-down analyses indicated that OsRad6 binds to OsSgt1, and transcripts of both OsRad6 and OsSgt1 were found to be strongly expressed only in the proliferating tissues such as the shoot apical meristem, suggesting that their expression is cell cycle-dependent. The amount of the Rad6 mRNA in cultured cells increased rapidly after division was halted, and mRNA levels of Rad6 and Sgt1 were induced by UV- and DNA-damaging agents such as MMS or H(2)O(2). The Rad6 pathway for repair or the proteasome system may thus require Sgt1 as ubiquitin-conjugating enzyme.

Degradation of proliferating cell nuclear antigen by 26S proteasome in rice (Oryza sativa L.).

To study whether metabolic control of proliferating cell nuclear antigen (PCNA) during the cell cycle is similar to that of associated protein factors, two-hybrid analysis with PCNA from rice (Oryza sativa L. cv. Nipponbare) was performed. PCNA interacted with rice Rpt6, which is the ATPase subunit of 26S proteasome, both in vitro and in vivo, and the degradation of PCNA was disrupted by the proteasome in vivo. The tissue-specific expression pattern of the transcripts of Rpt6 and PCNA suggested that the rice proteasome played important roles in DNA replication involving PCNA. These findings indicate a proteasome-dependent degradation of PCNA.

Cell-cycle-related variation in proteins in suspension-cultured rice cells.

To understand the cell cycle process in plants, we searched for proteins that quantitatively change during the cell cycle in suspension-cultured rice ( Oryza sativa L.) cells. The proteins were analyzed by a two-dimensional polyacrylamide gel electrophoresis image-analysis system. We detected 11 proteins that quantitatively changed during the cell cycle, among which beta-tubulins and a calreticulin-like protein were identified. The amounts of beta-tubulin proteins were low in the M phase and high in the G1 phase. In contrast, mRNAs for two of the three types of beta-tubulin were high in the M phase of the cell cycle. The addition of protease inhibitors MG132 or E64d to the cells decreased the beta-tubulin proteins during 24 h, suggesting that beta-tubulin proteins are degraded in vivo by proteases other than those whose activities are inhibited by MG132 or E64d.

Functional characterization of two flap endonuclease-1 homologues in rice.

Flap endonuclease-1 (FEN-1) is an important enzyme involved in DNA replication and repair. Previously, we isolated and characterized a complementary DNA (cDNA) from rice (Oryza sativa) encoding a protein which shows homology with the eukaryotic flap endonuclease-1 (FEN-1). In this report, we found that rice (O. sativa L. cv. Nipponbare) possessed two FEN-1 homologues designated as OsFEN-1a and OsFEN-1b. The OsFEN-1a and OsFEN-1b genes were mapped to chromosome 5 and 3, respectively. Both genes contained 17 exons and 16 introns. Alignment of OsFEN-1a protein with OsFEN-1b protein showed a high degree of sequence similarity, particularly around the N and I domains. Northern hybridization and in situ hybridization analysis demonstrated preferential expression of OsFEN-1a and OsFEN-1b in proliferating tissues such as the shoot apical meristem or young leaves. The levels of OsFEN-1a and OsFEN-1b expression were significantly reduced when cell proliferation was temporarily halted by the removal of sucrose from the growth medium. When the growth-halted cells began to regrow following the addition of sucrose to the medium, both OsFEN-1a and OsFEN-1b were again expressed at high level. These results suggested that OsFEN-1a and OsFEN-1b are required for cell proliferation. Functional complementation assay suggested that OsFEN-1a cDNA had the ability to complement Saccharomyces cerevisiae rad27 null mutant. On the other hand, OsFEN-1b cDNA could not complement the rad27 mutant. The roles of OsFEN-1a and OsFEN-1b in plant DNA replication and repair are discussed.

Interaction between proliferating cell nuclear antigen and JUN-activation-domain-binding protein 1 in the meristem of rice, Oryza sativa L.

The eukaryotic polymerase processivity factor, proliferating cell nuclear antigen (PCNA), interacts with many cell cycle-regulator proteins and with proteins involved in the mechanisms of DNA replication and repair. In the present study using two-hybrid analysis with PCNA from rice, Oryza sativa L. cv. Nipponbare (OsPCNA), we found that OsPCNA interacted in vitro and in vivo with rice JUN-activation-domain-binding protein 1 (OsJab1), which is known as COP9/signalsome subunit 5. Both OsPCNA and OsJab1 transcripts were expressed strongly in the shoot apical meristem and weakly in young leaves, flag leaves, ears, roots and root tips. No expression was detected in the mature leaves. The OsPCNA and OsJab1 proteins were expressed and accumulated mostly in the shoot apical meristem and ears, suggesting that OsJab1 is involved in cell proliferation in cooperation with OsPCNA. The role of OsPCNA with OsJab1 in plant DNA proliferation is discussed.

Cell cycle function of a rice B2-type cyclin interacting with a B-type cyclin-dependent kinase.

Cyclin-dependent kinases (CDKs) are involved in the control of cell cycle progression. Plant A-type CDKs are functional homologs of yeast Cdc2/Cdc28 and are expressed throughout the cell cycle. In contrast, B-type CDK (CDKB) is a family of mitotic CDKs expressed during the S/M phase, and its precise function remains unknown. Here, we identified two B2-type cyclins, CycB2;1 and CycB2;2, as a specific partner of rice CDKB2;1. The CDKB2;1-CycB2 complexes produced in insect cells showed a significant level of kinase activity in vitro, suggesting that CycB2 binds to and activates CDKB2. We then expressed green fluorescent protein (GFP)-fused CDKB2;1 and CycB2;2 in tobacco BY2 cells to investigate their subcellular localization during mitosis. Surprisingly, the fluorescence signal of CDKB2;1-GFP was tightly associated with chromosome alignment as well as with spindle structure during the metaphase. During the telophase, the signal was localized to the spindle midzone and the separating sister chromosomes, and then to the phragmoplast. On the other hand, the CycB2;2-GFP fluorescence signal was detected in nuclei during the interphase and prophase, moved to the metaphase chromosomes, and then disappeared completely after the cells passed through the metaphase. Co-localization of CDKB2;1-GFP and CycB2;2-GFP on chromosomes aligned at the center of the metaphase cells suggests that the CDKB2-CycB2 complex may function in retaining chromosomes at the metaphase plate. Overexpression of CycB2;2 in rice plants resulted in acceleration of root growth without any increase in cell size, indicating that CycB2;2 promoted cell division probably through association with CDKB2 in the root meristem.

Rice UV-damaged DNA binding protein homologues are most abundant in proliferating tissues.

Ultraviolet-damaged DNA binding protein (UV-DDB) is an important factor involved in DNA repair. To study the role of UV-DDB, we attempted to obtain the cDNA and the protein of a plant UV-DDB. We succeeded in isolating both genes for UV-DDB subunits from rice (Oryza sativa cv. Nipponbare), designated as OsUV-DDB1 and OsUV-DDB2. OsUV-DDB2 (65 kDa) was much larger than human UV-DDB2, but immunoprecipitation and gel mobility shift assay suggested that OsUV-DDB2 is a plant counterpart of UV-DDB2. The transcripts were expressed in proliferating tissues such as the meristem, but were detected at only low levels in the mature leaves, although the leaves are strongly exposed to UV. These transcripts were induced in the meristem after UV-irradiation. The expression levels of OsUV-DDB were significantly reduced when cell proliferation was temporarily halted. These results indicated that the level of OsUV-DDB expression is correlated with cell proliferation, and its expression may be required mostly for DNA repair in DNA replication.

OsSEND-1: a new RAD2 nuclease family member in higher plants.

A novel endonuclease, a new member of the RAD2 nuclease family, has been identified from the higher plant, rice (Oryza sativa L. cv. Nipponbare), and designated as OsSEND-1. The open reading frame of the OsSEND-1 cDNA encoded a predicted product of 641 amino acid residues with a molecular weight of 69.9 kDa. The encoded protein showed a relatively high degree of sequence homology with the RAD2 nuclease family proteins, especially RAD2 nuclease, but it differed markedly from FEN-1, XPG or HEX1/EXO1. The N- and I-domains in the family were highly conserved in the OsSEND-1 sequence. The protein was much smaller than XPG, but larger than HEX1/EXO1 and FEN-1. The genome sequence was composed of 14 exons, and was localized at the almost terminal region of the short arm of chromosome 8. Northern blotting and in situ hybridization analyses demonstrated preferential expression of OsSEND-1 mRNA in proliferating tissues such as meristem. The mRNA level of OsSEND-1 was induced by UV and DNA-damaging agent such as MMS or H2O2, indicating that OsSEND-1 has some roles in the repair of many types of damaged DNA. The recombinant peptide showed endonuclease activity.

Characterization of all the subunits of replication factor C from a higher plant, rice (Oryza sativa L.), and their relation to development.

Replication factor C (RFC), which is composed of five subunits, is an important factor involved in DNA replication and repair mechanisms. Following previous studies on the RFC3 homologue from rice (Oryza sativa L. cv. Nipponbare) (OsRFC3), we succeeded in isolating and characterizing one large and three small subunits of RFC homologues from the same rice species and termed them OsRFC1, OsRFC2, OsRFC4 and OsRFC5. The plant was found to have all RFC subunits known in yeasts, human and other eukaryotes. The open reading frames of OsRFCs encoded a predicted product of 1021 amino acid residues with a molecular mass of 110.8 kDa for OsRFC1, 339 amino acid residues with a molecular mass of 37.4 kDa for OsRFC2, 335 amino acid residues with a molecular mass of 36.8 kDa for OsRFC4, and 354 amino acid residues with a molecular mass of 40.5 kDa for OsRFC5. All the OsRFC subunits have highly conserved amino acid motifs among RFC proteins, RFC box, and an unrooted phylogenetic tree shows each OsRFC subunit belongs to each RFC subunit group. These subunits showed differences in their expression patterns among tissues. The transcripts of OsRFCs were expressed strongly in the proliferating tissue, the shoot apical meristem (SAM), and very weakly in the mature leaves which have no proliferating tissues. However, in young leaves and flag leaves, tissue-specific expression of OsRFC3 and OsRFC4 was shown. On the other hand, cell cycle arrest by cell cycle inhibitors resulted in significant differences in OsRFC expression patterns. These results suggest the functional differences of each OsRFC subunit in tissues and the plant cell cycle. The roles of these molecules in plant DNA replication and DNA repair are discussed.

Characterization of DNA polymerase delta from a higher plant, rice (Oryza sativa L.).

DNA polymerase delta (pol delta), which is comprised of at least two essential subunits, is an important enzyme involved in DNA replication and repair. We have cloned and characterized both the catalytic and small subunits of pol delta from rice (Oryza sativa L. cv. Nipponbare). The open reading frames of OsPoldelta1 and delta2 encoded a predicted product of 1105 amino acid residues with a molecular weight of 124 kDa for OsPoldelta1, and of 429 residues with a molecular weight of 48 kDa for OsPoldelta2. Northern blotting analysis indicated that OsPoldelta1 and delta2 transcripts were expressed strongly in proliferating tissues such as shoot apical meristem. The expression patterns of both subunits in the organs were slightly different. Therefore, we analyzed the spatial distribution pattern of OsPoldelta1 transcripts by in situ hybridization. In the shoot apex, OsPoldelta1 mRNA was abundant in the shoot apical meristem. In the roots, the OsPoldelta1 transcript accumulated at high levels in the root apical meristem. In mature leaves, OsPoldelta1 was induced after UV irradiation, but OsPoldelta2 was not. The amounts of the OsPoldelta1 and delta2 mRNAs in the rice cells changed rapidly during cell proliferation. These results indicated that the levels of OsPoldelta expression are markedly correlated with cell proliferation, and that some of OsPoldelta might have special roles in the leaves.

Cell-cycle dependent dynamic change of 26S proteasome distribution in tobacco BY-2 cells.

The 26S proteasome is known to play pivotal roles in cell-cycle progression in various eukaryotic cells; however, little is known about its role in higher plants. Here we report that the subcellular distribution of the 26S proteasome is dynamically changed in a cell-cycle dependent manner in tobacco BY-2 cells as determined by immunostaining with anti-Rpn10 (a regulatory PA700 subunit) and anti-20S catalytic proteasome antibodies. The 26S proteasome was found to localize not only in nuclear envelopes and mitotic spindles but also in preprophase bands (PPBs) and phragmoplasts appearing in G(2) and M phases, respectively. MG132, a proteasome inhibitor, exclusively caused cell-cycle arrest not only at the metaphase but also the early stage of PPB formation at the G(2) phase and the collapse of the phragmoplast, which seems to be closely related to proteasome distribution in the cells.

Spatial distribution of the 26S proteasome in meristematic tissues and primordia of rice (Oryza sativa L.).

The 26S proteasome is known to play central roles in the growth of many eukaryotes. However, little is known regarding its distribution in higher plants. Here, we report the spatial distribution pattern of Rpn3 (a regulatory PA700 subunit) and C2 (a subunit of the 20S proteasome) in rice ( Oryza sativa L.) seedlings as determined by in situ hybridization. The transcripts were abundantly co-expressed in the apical and marginal meristems of shoots and roots. Interestingly, these transcripts also accumulated in the leaf and ligule primordia of the shoot apex. Our results suggest that the 26S proteasome is spatially distributed among various tissues and may be involved not only in cell division but also in organ formation in higher plants.