Research Area

Cell Cycle Markers


Introduction of cell cycle markers

The cell cycle refers to the whole process that the cell undergoes from the completion of one split to the end of the next split and is divided into two phases: the interphase and the split phase. Life is a continuous process that passes from one generation to the next, so it is a process of constantly updating. The life of a cell begins with the division of its parent cell, the formation of its daughter cells, or the death of the cell itself. The formation of daughter cells is usually a sign of the end of a cell division, which refers to the process that occurs from the time when a cell divides to form a daughter cell until the next cell divides to form a daughter cell. In this process, the genetic material of the cell is replicated and equally distributed to the two daughter cells. In recent years, cell cycle markers such as minichromosome maintenance protein, geminin, cyclinB1 and serine 10 phosphorylation histone 3 have become hotspots in the research of diseases such as tumors.

Cell cycle markers family members and their functions respectively

Minichromosome maintenance protein (MCM) is a family of highly conserved proteins originally discovered in yeast. Its members include MCM2, MCM3, MCM4 (CDC54), MCM5, MCM6 (MIS5), MCM7 (CDC47), etc. These proteins can act as a DNA replication regulator, so it is a molecule necessary for eukaryotic DNA replication. Members of the MCM protein family have different structural compositions, but each has a characteristic ATPase region of approximately 200 amino acid sequences. In addition, MCM2, MCM4, MCM6, and MCM7 also contain a zinc finger region that plays an important role between DNA and protein. The expression of MCM in cells is periodic. When cells enter the G0 phase, most of MCM is not expressed, or its expression level is the lowest. Entering G1, MCM combines with chromatin and reaches its highest value at the end of G1. After entering the S phase, due to the co-regulation of CDK and DDK, MCM is dissociated from the chromatin until the free microchromosomes appear in the M phase, so it is expressed in the G1-M phase. Since the MCM2 study in the MCM protein family has been intensive in recent years, the application of MCM2 in malignant tumors will be highlighted later. Geminin is a small molecule multifunctional protein located in the nucleus. It has complex structural and functional regions and plays an extremely important role in cell proliferation, embryonic development, and tumor formation. Geminin was cloned in Xenopus egg cells in 1998 and confirmed to be present in human cells. It has two isomers, Geminin H and Geminin L, which contain 219 and 216 amino acids, respectively. Geminin is capable of negatively regulating CDT1, a key factor in DNA replication initiation, and blocks DNA replication during the cell cycle to induce information on pre-replication complexes. Geminin is mainly present in cells entering the cell cycle, and it is not expressed in quiescent or hypoplastic cells. It is the initial signal for the G1 phase to S phase transformation. At the initial phase of the S phase, Geminin begins to appear in the nucleus, as the cell cycle progresses, and interferes with the stability of Pre-RC (pre-replication complex) by binding to CDT1, preventing MCM from loading onto the chromosome, causing DNA replication to stop; it accumulates in the G2 phase. It also reached a peak in the M phase, and as the activity of the late-promoting complex (APC) increased, the geminingene "destruction box" mutation was induced to degrade before the next G1 phase. Since tumor cells follow the cell cycle pattern during proliferation as well as normal cells, many studies have recently used Geminin as a cell cycle S/G2/M phase marker to label malignant tumors and evaluate prognosis. CyclinB1 is the first discovered cyclin, and the coding gene is located at 5q12. It is a cyclin involved in the regulation of cell proliferation, and its protein expression level changes with cell cycle progression. It binds to CDK (cell cycle-dependent kinase) and has a phosphorylation target protein action. When the cell cycle enters the G1 phase, the expression level of cyclinB1 protein is very low. As the cell cycle progresses, its expression level gradually increases to S and G2 and forms MPF enzyme complex with CDK1 until G2 phase. Under the action of CDC25C, CDK1 dephosphorylation releases the inhibition of its activity, thereby activating MPF; this activation process continues until the end of mitosis. CyclinB1 rapidly degraded and disappeared, MPF was inactivated, and the cells exited the mitosis phase and entered the next cell cycle. CyclinB1 plays an important role in regulating the cell cycle in G2/M phase. Some studies have recently used it as a diagnostic marker. Serine 10-phosphorylation histone H3 (H3S10ph) is a DNA-binding protein that is essential for cell cycle, located at the tail of histone H3. H3S10ph is a mitotic marker of unknown function. Studies have shown that it is formed by the phosphorylation of histone H3, a serine residue Ser10 during mitosis. During mitosis, H3S10ph is catalyzed by Aurora B kinase (Aur-B), which first appears in the periphery of the G2 late core, peaks in the mid-term as the cell cycle progresses, and extends to all parts of the chromosome. The mitosis of the cells enters the late stages, and H3S10ph appears in the central part of the spindle until the end of mitosis, and H3S10ph dephosphorylates. This means that H3S10ph persists throughout mitosis and plays an important role in chromosome aggregation and segregation in M phase. Since the large occurrence of H3S10ph is significantly correlated with the late G2 phase to the M phase, it has been used as a specific marker for M phase in some studies targeting the mitosis of tumor cells.

Functions of cell cycle markers

Gonzalez et al studied the expression of MCM2 and Ki67 in 347 breast cancers, using tissue microarray and immunohistochemistry, and found that MCM2 is more frequently expressed in breast cancer than the conventionally used proliferation marker Ki67 (P < 0. 000 1), and the expression of MCM2 in 221 invasive breast cancers with tumor size (P = 0.002), mitotic index (P < 0.001), histological grade (P < 0. 0001) are relevant to the Nottingham Prognostic Index score (The NPI is used to determine prognosis following surgery for breast cancer. Its value is calculated using three pathological criteria: the size of the lesion; the number of involved lymph nodes; and the grade of the tumour) (P < 0.0001). Studies using 50% cut-off values found MCM2 and overall survival (P = 0. 000 7), healthy interval (P = 0. 000 2), regional recurrence (P = 0. 011), and distant metastasis (P = 0.001) relevant, and finally concluded that MCM2 as a prognostic marker combined with NPI can predict the outcome of breast cancer. The lower risk of death in patients with high MCM2 expression was significantly higher ( RR = 3. 389, 95% CI =1. 803 ~ 7.146, P < 0.001), and the cumulative survival rate was significantly lower (P =0. 001), and multivariate prognostic analysis showed that MCM2 expression was an independent prognostic factor for NSCLC (P=0.041). Analysis of the results suggests that MCM2 may be a cell proliferation marker superior to Ki67, which can be used to grade esophageal squamous cell carcinoma and to determine the degree of malignancy of esophageal squamous cell carcinoma. Liu et alused immunohistochemistry to label 108 cases of gallbladder adenocarcinoma, 15 cases of gallbladder polyps, 35 cases of chronic cholecystitis and 46 cases of adjacent tissues with MCM2 and TIP30 to screen for specific markers for early diagnosis. From the expression of immunohistochemistry, MCM2 expression in adenocarcinoma was higher than in the other three tissues. At the same time, the expression of MCM2 is significantly associated with poor differentiation, tumor size, lymph node metastasis and invasiveness of adenocarcinoma. Univariate Kaplan-Meier analysis suggested that increased expression of MCM2 (P = 0.006) was associated with a decrease in overall survival; multivariate cox regression analysis suggested that expression of MCM2 (P = 0.007) could be used as a prognosis for adenocarcinoma. Finally, it is concluded that the overexpression of MCM2 is closely related to the occurrence, development, biological behavior and prognosis of gallbladder adenocarcinoma. Shomoriet al. analyzed seven gastric cancer cell lines by western blot, analyzed 72 cases of gastric mucosal lesions and 128 cases of surgically resected advanced intestinal-type gastric cancer by immunohistochemistry, and identified the Geminin and Ki67 by immunofluorescence double labeling. After co-expression, Geminin was found in all cell lines, and low-grade and high-grade adenomas and intestinal adenocarcinomas was 3.9%, 10.5%, 18.6 %, 27.2%; Immunofluorescence double labeling revealed that both Geminin and Ki67 were co-expressed in both normal and tumor cells. According to the staging of the International Anticancer Organization (UICC), Geminin is significantly associated with the N phase. Univariate Cox regression analysis indicated that the total survival of tumors from stage I to IV was significantly associated with higher Geminin LI (related risk RR = 1.94; P = 0.04). It was concluded that the expression of Geminin may reflect the biological properties of tumors in the gastric mucosa and may be a prognostic marker for advanced intestinal-type gastric cancer. Ersvaer et al. used flow cytometry and confocal microscopy to study the expression of cyclinB1 in cells of patients with acute myeloid leukemia (AML). It was found that cyclinB1 was expressed in 42 cases, and abnormalities occurred in 32 of them. CyclinB1 is also continuously expressed in the cytoplasm during the next 14 days of in vitroculture, and cyclinB1-specific antibodies were present in 7 of the 65 AML patients who were not treated. Therefore, the expression of cyclinB1 occurs in many cytoplasmic abnormal AML cells, and a specific humoral immune response against cyclin B1 occurs in some untreated leukemia patients. The expression levels of cyclinB1 and proliferating cell nuclear antigen (PCNA) in 62 human gliomas and 14 normal brain tissues were detected by immunohistochemistry. The expression levels of cyclinB1 and proliferating cell nuclear antigen (PCNA) were observed and compared. The expression rate of cyclinB1 in normal brain tissue was 7.1% (1/14), and the positive rate and average marker index increased significantly with the increase of pathological grade of glioma (P < 0.05). Finally, it is concluded that the expression of cyclinB1 in human glioma is closely related to pathological grade and tumor cell proliferation activity, which may be a contributing factor to the malignant proliferation of tumor.

Reference

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  3. Thomasova D, Anders H. Cell cycle control in the kidney. Nephrology, dialysis, transplantation: official publication of the European Dialysis and Transplant Association - European Renal Association. 2015, 30(10):1622.
  4. Krabbe L M, Margulis V, Lotan Y. Prognostic Role of Cell Cycle and Proliferative Markers in Clear Cell Renal Cell Carcinoma. Urologic Clinics of North America. 2016, 43(1):105.
  5. Yousaf J, Hills C, Dixit S, et al. Markers of cell division cycle in glioblastoma: significance in prediction of treatment response and patient prognosis. British Journal of Neurosurgery. 2013, 27(6):752-758.

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