Dysregulated cell cycle progression serves a crucial role in tumor development. Cell division cycle- associated 3 (CDCA3) is considered a trigger of mitotic entry; it is an important part of the S phase kinase-associated protein 1/Cullin/F-box ubiquitin ligase complex and mediates the destruction of mitosis-inhibitory kinase wee1. However, little is known about the role of CDCA3 in cancer, particularly colorectal cancer (CRC). The present study aimed to explore the biological and clinical significance of CDCA3 in CRC growth and progression. CDCA3 expression was significantly associated with tumor progression and poor survival. Overexpression of CDCA3 increased proliferation in LoVo CRC cells, whereas CDCA3 knockdown in SW480 CRC cells led to decreased proliferation,
Despite the known pathogenic risks and the development of progressive therapeutic strategies, colorectal cancer (CRC) has the fourth highest incidence rate among all cancers and the fifth highest incidence rate of tumor-related mortalities in China (
Aberrant cellular growth and division are common features in malignant cells, including CRC cell lines (
Cell division cycle-associated protein 3 (CDCA3) is referred to as a ‘trigger’ of mitotic entry, and has been reported to mediate cell cycle progression (
A total of 124 CRC and 124 (71 male; 53 female; age, 23-88 years) adjacent non-tumor colorectal tissue samples, including a total of 84 patients that were detected in
CDCA3 expression in 40 CRC tissues and 40 adjacent normal specimens were detected by IHC. Immunohistochemical staining was performed using a standard immunoperoxidase staining procedure. The tissues were fixed in 10% formalin for 48 h at room temperature. The paraffin-embedded tumor tissue sections (4
CRC cell lines SW480, LoVo, DLD-1, HCT116, Caco-2 and HT29, and the normal human colorectal epithelial cells NCM460 were maintained in our laboratory. All cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (Wisent, Inc. St. Bruno, QC, Canada), 100 U/ml penicillin and 100
Total RNA was extracted from ~100 mg tissues or cultured cells when they reached a density of 90% using TRIzol® Reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA), according to the manufacturer’s protocol. Total RNA was reverse transcribed into cDNA using the PrimeScript RT reagent kit (Takara Biotechnology Co., Ltd., Dalian, China). qPCR was performed in a 20
Small interfering (si)RNAs against CDCA3 (siCDCA3), E2F1 (siE2F1) and negative control (siNC) were designed and synthesized by Shanghai GenePharma Co., Ltd. (Shanghai, China). The sequences for the CDCA3 siRNAs were as follows: siRNA1-CDCA3, sense, 5′-GCAAUAGAUGGAAACCAA ATT-3′ and antisense, 5′-UUUGGUUUCCAUCUAUUGCTT-3′; siRNA2-CDCA3, sense, 5′-GAGUGAAGUAUUUGAAAC UTT-3′, antisense, 5′-AGUUUCAAAUACUCTT-3′; siRNA3- CDCA3, sense, 5′-GCUCUCCUACUCUUGGUAUTT-3′, antisense, 5′-AUACCAAGAGUAGGAGAGCTT-3′; siE2F1, sense, 5′-CCUGGAAACUGACCAUCAGTT-3′, antisense, 5′-CUGAUGGUCAGUUUCCAGGTT-3′; and siNC, sense, 5′-UUCUCCGAACGUGUCACGUTT-3′, antisense, 5′-ACGUGACACGUUCGGAGAATT-3′. siRNAs (5
For the CDCA3 overexpression assays, a mammalian expression plasmid pReceiver-M02-CDCA3 (CDCA3-OE; GeneCopoeia, Inc., Rockville, MD, USA) was designed to specifically express CDCA3 and used empty vectors (pReceiver-M02-control) as the control. Plasmids (5
shRNA- and shNC-SW480 cells were transiently transfected with pReceiver-M02-E2F1 (pR-E2F1) or pReceiver-M02-control (pR-contol) (GeneCopoeia, Inc.) as aforementioned. In addition, CDCA3-SOE- and control-SOE-LoVo cells were transiently transfected with si-E2F1 or si-NC (GenePharma Co., Ltd, Shanghai, China) following the above process.
For colony formation assays, 500 SW480 cells transfected with siRNA1-CDCA3 or siNC, and CDCA3-OE-LoVo or cells with empty vectors (control-OE), were plated into 6-well plates 48 h post-transfection. Following incubation at 37°C for 14 days, the clones were visible. Each well was washed with PBS three times, fixed with ethyl alcohol for 30 sec and stained with 0.05% crystal violet for 20 min at room temperature. After washing, the colonies (≥50 cells/colony) were counted using a Nikon TI-DH light microscope (Nikon Corporation, Tokyo, Japan) and images captured using a Canon DS126211 digital camera (Canon, Inc., Tokyo, Japan). In the later assays, shRNA- and shNC-SW480 cells transiently transfected with pReceiver-M02-E2F1 (pR-E2F1) or pReceiver-M02-control (pR-control) (GeneCopoeia, Inc.), as well as the CDCA3-SOE- and control-SOE-LoVo cells transiently transfected with si-E2F1 or si-NC (GenePharma Co., Ltd.), were plated into 6-well plates 48 h following transfection and treated as aforementioned.
The Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Kumamoto, Japan) assay was used to detect cell proliferation, according to the manufacturer’s protocol. Cells (2×103 cells/well) were seeded in 96-well plates with 100
The EdU assay kit (cat. no. C10310-3; Guangzhou RiboBio Co., Ltd., Guangzhou, China) was also used to measure cell proliferation. Briefly, cells were seeded (2×104 cells/well) into 24-well plates and cultured with DMEM for 24 h. Subsequently, cells were incubated with EdU (200
Following 48 h transfection, cells were digested with trypsin and centrifuged at room temperature for 5 min at 300 x g. Cells were washed carefully with PBS two times, fixed in 75% ethanol and stored at -20°C overnight. Subsequently, the cells were washed twice with PBS and stained with propidium iodide (PI) staining solution (500
For apoptosis, cells were stained with PI (10
Protein lysates were prepared from the cells when they reached a density of 90% by using a Radioimmunoprecipitation assay kit (Beyotime Institute of Biotechnology, Shanghai, China), according to the manufacturer’s protocols. The protein concentration was determined using the Bicinchoninic Acid Protein Assay kit (Beyotime Institute of Biotechnology). Proteins (40
RNA expression data (level 3) were downloaded from The Cancer Genome Atlas (TCGA;
Database for Annotation, Visualization and Integrated Discovery (DAVID) bioinformatics resources (
A total of 12 Balb/c nude male mice (age, 3-4 weeks; weight, 13-15 g) were purchased from the Animal Center of NJMU. The 12 mice were randomly divided into two groups (n=6 per group) and were maintained at 20-26°C, 40-70% humidity, ammonia concentration <14 mg/m3, with a 12-h light/dark cycle and received 5 g food and 100 ml water per 100 g body weight per day. A total of 2×106 stably transfected cells (shCDCA3 or shNC) were randomly and subcutaneously injected into each mouse in their right armpit. Bidimensional tumor measurements were taken with Vernier calipers every 4 days. All mice were sacrificed 4 weeks later and the tumors were surgically removed. Implanted tumor volume was calculated according to the following the formula: Volume = (width2 x length)/2. Following sacrifice, tumor tissues were prepared for IHC as aforementioned and stained with rabbit monoclonal anti-Ki-67 (1:500; cat. no. ab92742; Abcam). The staining intensity was visually scored as follows (
Data are expressed as the mean ± standard deviation. GraphPad Prism 5.0 software (GraphPad Software Inc., La Jolla, CA, USA) and the Statistical Program for Social Sciences 20.0 (IBM Corp., Armonk, NY, USA) were used to analyze the data. The clinical features were analyzed using χ2 test. The Wilcoxon rank-sum test was used to compare CDCA3 protein expression in CRC and adjacent normal tissue. Student’s t-test was used to compare the treated and control groups. One-way analysis of variance and the least-significant difference post hoc test were used to compare data sets containing multiple groups. Pearson’s correlation test was used to examine gene co-expressions; r≥0.5 was considered to indicate a relatively strong correlation. Cumulative survival analysis was assessed using the Kaplan-Meier method followed by log-rank test. P<0.05 was considered to indicate a statistically significant difference.
To investigate the functional role of CDCA3 in CRC, the mRNA expression levels of CDCA3 were compared in 84 pairs of CRC and adjacent normal tissues using RT-qPCR, which indicated that CDCA3 expression was significantly higher in CRC tissues compared with adjacent normal tissue (P<0.001;
To further investigate the functional role of CDCA3 in CRC cells, CDCA3 mRNA and protein expression levels were examined in six CRC cell lines using RT-qPCR and western blotting (
CDCA3 is a known ‘trigger’ of mitosis entry (
To explore the possible roles of CDCA3 in influencing CRC cell proliferation, cell cycle distribution and apoptotic cell fraction were examined. To constitutively suppress or overexpress CDCA3, four stable cell lines were constructed: shCDCA3-SW480 and shNC-SW480, as well as CDCA3-SOE-LoVo and Control-SOE-LoVo. The efficiency of knockdown or overexpression was confirmed by western blotting (
To further explore the mechanism how CDCA3 mediates p21 expression, correlation analysis was performed using the R software (version 3.4.0) through analyzing the data obtained from TCGA. A total of 355 genes whose correlation coefficients were ≥0.5 were selected. Subsequently, GO analysis and a KEGG pathway analysis were examined using DAVID to determine whether any GO terms or KEGG pathways were enriched. As observed from the gene enrichment of GO and KEGG analysis, the cell cycle is the largest group in both analyses (
To verify this hypothesis, E2F1 expression in shCDCA3- SW480 and CDCA3-SOE-LoVo cells was examined using RT-qPCR and western blotting. The results demonstrated that E2F1 mRNA and protein expression levels were notably decreased shCDCA3-SW480 cells and increased in the CDCA3-SOE-LoVo cells (
Additional experiments were conducted to determine whether E2F1 was able to reverse the inhibition of proliferation induced by CDCA3 downregulation. The colony formation assay and CCK-8 assay indicated that overexpression of CDCA3 promoted cell proliferation in LoVo cells; however, this promotion of cell proliferation was inhibited by si-E2F1 (
To further confirm the effects of CDCA3
CDCA3 is part of the SCF E3 ligase complex that regulates mitotic entry (
p21, one of the key regulators of cell cycle progression at the G1/S transition, serves a crucial role in tumorigenesis (
However, there are still many topics that remain to be explored. For example, the effects of CDCA3 on E2F1 and the mechanisms involved need further investigation. In addition, considering that CDCA3 expression is significantly associated with lymph node invasion in CRC,
The present study was funded by Jiangsu Key Medical Discipline- General Surgery (grant no. ZDXKA2016005).
The data sets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
YS conceptualized and designed the research. WQ performed experiments, analyzed and interpreted the results, made figures and wrote the paper. ZZ, WP and JL performed experiments and analyzed data. JL provided patient tissues. QG, DJ, QW and YZ helped design the experimental studies and edited the manuscript. BJ, SW and DZ interpreted the results and wrote the manuscript. All authors have read and approved the manuscript.
The present study was approved by the Research Ethics Committee of The First Affiliated Hospital of Nanjing Medical University and written informed consent was obtained from all patients prior to enrolment in the study.
Not applicable.
The authors declare that they have no competing interests.
The authors would like to thank the faculty and staff of the Department of General Surgery (First Affiliated Hospital of Nanjing Medical University, Jiangsu, China) for providing language and technology support.
Expression of CDCA3 in CRC tissues and cell lines and Kaplan-Meier survival analysis. (A) Relative CDCA3 mRNA expression levels in 84 pairs of CRC tissues and adjacent normal tissues detected by RT-qPCR. (B) Kaplan-Meier survival analysis of 84 patients with CRC based on high or low CDCA3 expression levels. (C) CDCA3 protein expression was examined by immunohistochemistry in 40 CRC tissues and 21 adjacent normal specimens. (D and E) CDCA3 mRNA and protein expression levels were examined in six CRC cell lines using (D) RT-qPCR and (E) western blotting. Data are expressed as the mean ± standard deviation; ***P<0.001. CDCA3, cell division cycle-associated protein 3; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.
Expression of CDCA3 in CRC cells in loss or gain of function assays. (A) CDCA3 mRNA expression levels were examined by reverse transcription- quantitative polymerase chain reaction in SW480 cells following transfection with different siRNAs against CDCA3 or siNC. (B-E) SW480 cells were transfected with siRNA1-CDCA3, and LoVo cells were transfected with CDCA3 overexpression vector and used to examine: (B) CDCA3 protein expression levels by western blotting; cell proliferation by (C) Cell counting kit-8 assays and (D) EdU staining assays; and (E) colony-formation ability. Representative images are presented in B, D and E. Data are presented as the mean ± standard deviation from three independent experiments; *P<0.05, **P<0.01 and ***P<0.001. CDCA3, cell division cycle-associated protein 3; CRC, colorectal cancer; EdU, 5-ethynyl-2’-deoxyuridine; NC, negative control; pR, pReceiver-M02; si, small interfering RNA.
CDCA3 influences G1/S phase transition of the cell cycle by regulating p21 expression. (A) The efficiency of knockdown or overexpression was confirmed by western blotting detection of CDCA3 protein expression levels. (B) shCDCA3 notably increased the number of cells in G1 phase compared with the shNC control. (C) Overexpression of CDCA3 promoted the G1/S transition in LoVo cell lines. (D) Key regulators of the G1 phase were evaluated by western blotting. Data are presented as the mean ± standard deviation; *P<0.05 and **P<0.01. CDCA3, cell division cycle-associated protein 3; CDK2, cyclin-dependent kinase 2; NC, negative control; sh, short hairpin RNA.
E2F1 may be a potential downstream target of CDCA3. (A) Gene ontology analysis of the selected 355 genes. (B) Kyoto Encyclopedia of Genes and Genomes pathway analysis of the selected 355 genes. (C) mRNA expression levels of 29 genes in shCDCA3-SW480 and shNC-SW480 cells. (D) E2F1 expression was detected by RT-qPCR and western blotting in SW480 and LoVo cells, respectively. (E) E2F1 expression levels were measured by RT-qPCR and western blotting following downregulation of CDCA3 in SW480 and upregulation of CDCA3 in LoVo cells. Results are presented as the mean ± standard deviation; *P<0.05, **P<0.01 and ***P<0.001. BUB1, BUB1 mitotic checkpoint serine/threonine kinase; BUB1B, BUB1 mitotic checkpoint serine/threonine kinase B; BUB3, BUB3 mitotic checkpoint protein; CCN, cyclin; CDC20, cell division cycle; CDCA3, cell division cycle-associated protein 3; CDK, cyclin-dependent kinase; CHEK, checkpoint kinase; ESPL1, extra spindle pole bodies-like 1, separase; MAD2L1, mitotic arrest deficient 2-like 1; MCM, minichromosome maintenance complex component; NC, negative control; PKMYT1, protein kinase, membrane-associated tyrosine/threonine 1; PLK1, polo-like kinase 1; PTTG1, pituitary tumor-transforming 1; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; sh, short hairpin RNA; si, small interfering RNA; TTK, TTK protein kinase.
CDCA3 mediates p21-dependent proliferation by regulating E2F1 expression. (A) p21 mRNA expression was detected in the indicated stable cell lines transfected with pR-E2F1-overexpression or pR-Control vector. (B) Western blotting was used to detect the protein expression levels of CDCA3, E2F1 and p21 following the various treatments. (C and D) Representative images of colony formation and results from cell counting kit-8 assays. Data are presented as the mean ± standard deviation; **P<0.01 and ***P<0.001. CDCA3-SOE- and Control-SOE-LoVo cells represent the relatively stable overexpression of CDCA3 in LoVo cells. CDCA3, cell division cycle-associated protein 3; NC, negative control; pR, pReceiver-M02; sh, short hairpin RNA; si, small interfering RNA.
Downregulation of CDCA3 inhibits CRC tumorigenesis
Relationship between CDCA3 expression and the clinicopathological characteristics of patients with CRC.
Characteristics | n | CDCA3 expression level
|
P-value (χ2) | |
---|---|---|---|---|
Low |
High | |||
Age (years) | ||||
<60 | 27 | 16 | 11 | 0.243 |
≥60 | 57 | 26 | 31 | |
Sex | ||||
Male | 49 | 23 | 26 | 0.507 |
Female | 35 | 19 | 16 | |
Tumor diameter (cm) | ||||
<5 | 47 | 31 | 16 | 0.001 |
≥5 | 37 | 11 | 26 | |
TNM stage | ||||
I/II | 43 | 26 | 17 | 0.049 |
III/IV | 41 | 16 | 25 | |
Lymph node invasion | ||||
Negative | 46 | 28 | 18 | 0.028 |
Positive | 38 | 14 | 24 | |
Depth of invasion | ||||
T1 + T2 | 21 | 14 | 7 | 0.078 |
T3 + T4 | 63 | 28 | 35 | |
Distant metastasis | ||||
Negative | 73 | 38 | 35 | 0.332 |
Positive | 11 | 4 | 7 | |
Primary tumor site | ||||
Colon | 40 | 18 | 22 | 0.382 |
Rectum | 44 | 24 | 20 |
P<0.001;
P<0.05. CDCA3, cell division cycle-associated protein 3; CRC, colorectal cancer; TNM, tumor, necrosis and metastasis.
Statistical analysis of CDCA3 protein expression in CRC and adjacent normal tissues.
Tissue | n | Total score
|
P-value | |||
---|---|---|---|---|---|---|
− | + | ++ | +++ | |||
CRC | 40 | 8 | 10 | 19 | 3 | 0.003 |
Adjacent normal tissue | 40 | 16 | 16 | 7 | 1 |
CDCA3, cell division cycle-associated protein 3; CRC, colorectal cancer.
Primer sequences used for reverse transcription- quantitative polymerase chain reaction.
Gene | Sequence (5′→3′) |
---|---|
BUB1 | F: AAATGACCCTCTGGATGTTTGG |
R: GCATAAACGCCCTAATTTAAGCC | |
BUB1B | F: AAATGACCCTCTGGATGTTTGG |
R: GCATAAACGCCCTAATTTAAGCC | |
BUB3 | F: GGTTCTAACGAGTTCAAGCTGA |
R: GGCACATCGTAGAGACGCAC | |
CCNA2 | F: CGCTGGCGGTACTGAAGTC |
R: GAGGAACGGTGACATGCTCAT | |
CCNB1 | F: TCGCATCAAACTCTCTGGCTA |
R: TGAGCGACTAAACTCACCACT | |
CCNB2 | F: CCGACGGTGTCCAGTGATTT |
R: TGTTGTTTTGGTGGGTTGAACT | |
CCNE1 | F: AAGGAGCGGGACACCATGA |
R: ACGGTCACGTTTGCCTTCC | |
CDCA3 | F: TGGTATTGCACGGACACCTA |
R: TGTTTCACCAGTGGGCTTG | |
CDC20 | F: GCACAGTTCGCGTTCGAGA |
R: CTGGATTTGCCAGGAGTTCGG | |
CDC25A | F: GTGAAGGCGCTATTTGGCG |
R: TGGTTGCTCATAATCACTGCC | |
CDC25C | F: TCTACGGAACTCTTCTCATCCAC |
R: TCCAGGAGCAGGTTTAACATTTT | |
CDC45 | F: TTCGTGTCCGATTTCCGCAAA |
R: TGGAACCAGCGTATATTGCAC | |
CDC6 | F: GCCGAACTAGAACAGCATCTT |
R: GGGCTGGTCTAATTTTTCCTGC | |
CDK1 | F: TGGGAAGTTGGTAGCTCTGAA |
R: CCAGGGTGCTTGTCCATGTA | |
CDK2 | F: TGTTTAACGACTTTGGACCGC |
R: CCATCTCCTCTATGACTGACAGC | |
CDK4 | F: GGGGACCTAGAGCAACTTACT |
R: CAGCGCAGTCCTTCCAAAT | |
CHEK1 | F: ATATGAAGCGTGCCGTAGACT |
R: TGCCTATGTCTGGCTCTATTCTG | |
CHEK2 | F: TCTCGGGAGTCGGATGTTGAG |
R: CCTGAGTGGACACTGTCTCTAA | |
E2F1 | F: ACGCTATGAGACCTCACTGAA |
R: TCCTGGGTCAACCCCTCAAG | |
E2F2 | F: CGTCCCTGAGTTCCCAACC |
R: GCGAAGTGTCATACCGAGTCTT | |
ESPL1 | F: CCGCCTTGAAGGAGTTCCTG |
R: GGGGTAGACACTAAGTAGCCAT | |
GAPDH | F: GTGGACATCCGCAAAGAC |
R: AAAGGGTGTAACGCAACTA | |
MAD2L1 | F: GAGAAGTCCGAAGAAACTCACG |
R: CCGAAGCGTTGAGAGGTTCC | |
MCM2 | F: ATGGCGGAATCATCGGAATCC |
R: GGTGAGGGCATCAGTACGC | |
MCM3 | F: TCTAAGCCGCCATTTCGATTT |
R: AAGACGCTGGAAAGCTGGATA | |
MCM6 | F: GAGGAACTGATTCGTCCTGAGA |
R: CAAGGCCCGACACAGGTAAG | |
MCM7 | F: CCTACCAGCCGATCCAGTCT |
R: CCTCCTGAGCGGTTGGTTT | |
PKMYT1 | F: CATGGCTCCTACGGAGAGGT |
R: ACATGGAACGCTTTACCGCAT | |
p21 | F: TGCAACTACTACAGAAACTGCTG |
R: CAAAGTGGTCGGTAGCCACA | |
PLK1 | F: CAGTCACTCTCCGCGACAC |
R: GAGTAGCCGAATTGCTGCTG | |
PTTG1 | F: ACCCGTGTGGTTGCTAAGG |
R: ACGTGGTGTTGAAACTTGAGAT | |
TTK | F: GTGGAGCAGTACCACTAGAAATG |
R: CCCAAGTGAACCGGAAAATGA |
BUB1, BUB1 mitotic checkpoint serine/threonine kinase; BUB1B, BUB1 mitotic checkpoint serine/threonine kinase B; BUB3, BUB3 mitotic checkpoint protein; CCN, cyclin; CDC20, cell division cycle; CDCA3, cell division cycle-associated protein 3; CDK, cyclin-dependent kinase; CHEK, checkpoint kinase; ESPL1, extra spindle pole bodies-like 1, separase; MAD2L1, mitotic arrest deficient 2-like 1; MCM, minichromosome maintenance complex component; PKMYT1, protein kinase, membrane-associated tyrosine/threonine 1; PLK1, polo-like kinase 1; PTTG1, pituitary tumor-transforming 1; TTK, TTK protein kinase; F, forward; R, reverse.
Correlation between the expression of CDCA3 and the other 29 genes.
Gene symbol | P-value | R-value | Gene symbol | P-value | R-value |
---|---|---|---|---|---|
BUB1 | 4.73E-32 | 0.57 | CHEK1 | 1.23E-33 | 0.59 |
BUB1B | 5.99E-30 | 0.56 | CHEK2 | 1.67E-33 | 0.58 |
BUB3 | 2.20E-26 | 0.53 | E2F1 | 1.28E-40 | 0.63 |
CCNA2 | 2.80E-51 | 0.69 | E2F2 | 4.56E-33 | 0.58 |
CCNB1 | 1.14E-55 | 0.71 | ESPL1 | 4.82E-40 | 0.63 |
CCNB2 | 1.13E-52 | 0.70 | MAD2L1 | 5.38E-43 | 0.65 |
CCNE1 | 5.87E-27 | 0.53 | MCM2 | 1.73E-30 | 0.56 |
CDC20 | 1.18E-56 | 0.72 | MCM3 | 7.56E-26 | 0.52 |
CDC25A | 7.50E-46 | 0.66 | MCM6 | 8.27E-26 | 0.52 |
CDC25C | 1.15E-52 | 0.70 | MCM7 | 1.42E-33 | 0.58 |
CDC45 | 1.13E-49 | 0.68 | PKMYT1 | 3.15E-51 | 0.69 |
CDC6 | 1.09E-32 | 0.58 | PLK1 | 4.59E-56 | 0.71 |
CDK1 | 2.73E-62 | 0.74 | PTTG1 | 2.75E-60 | 0.73 |
CDK2 | 1.26E-31 | 0.57 | TTK | 1.58E-35 | 0.60 |
CDK4 | 4.78E-33 | 0.58 |
BUB1, BUB1 mitotic checkpoint serine/threonine kinase; BUB1B, BUB1 mitotic checkpoint serine/threonine kinase B; BUB3, BUB3 mitotic checkpoint protein; CCN, cyclin; CDC20, cell division cycle; CDK, cyclin-dependent kinase; CHEK, checkpoint kinase; ESPL1, extra spindle pole bodies-like 1, separase; MAD2L1, mitotic arrest deficient 2-like 1; MCM, minichromosome maintenance complex component; PKMYT1, protein kinase, membrane-associated tyrosine/threonine 1; PLK1, polo-like kinase 1; PTTG1, pituitary tumor-transforming 1; TTK, TTK protein kinase.