Current methods of studying of the proliferative activity in experiment

Abstract

Proliferation is an increasing of the body's tissues by cell multiplication by division. It may refer to the processes of different nature. Thus, cell proliferation underlies regenerative tissue neoplasms; proliferation observed in various hyperplasias; finally cell proliferation underlying tumor growth [Mitchison, 2003, Sulić at al., 2005]. Therefore, the identification and determining of the activity of proliferative processes is an important task of experimental and practical medicine.

The aim of this review was to analyze the contemporary scientific literature data about methods of determining of the proliferative activity, as well as the choice of an optimal method for its assessing in tissues with expected low proliferative activity.

There are many approaches to assess the proliferative activity. A number of proteins involved in cell cycle regulation has been opened last decades. They may serve as selectable markers of proliferating cells either in vivo, or in vitro. Determining of the proliferative activity is possible with using of different methods, such as counting of the mitotic index, enzyme immunoassay, immunoblotting, immunohistochemical analysis, PCR techniques. Consider some of them.

Mitotic index. Determining of the mitotic index is based on counting of the percentage of dividing cells of the total number of cells analyzed. The test tissue is treated with colchicine or its derivatives - substances that have properties to prevent the formation of microtubules, thereby preventing divergence of individual chromosomes in anaphase. After processing tissue the cell cycle stops in it, thereby enabling the identification of mitotic cells at the time of application of the drug [Прохорова и др., 2003]. The mitotic index (MI) calculated using the formula:

MI = (P+M+A+T)/N * 100,

where (Р+М+А+Т) is amount of cells at the stages of prophase, metaphase, anaphase and telophase, respectively, and N – the whole amount of cells analyzed.

The main disadvantages of this method are:

¾              low specificity;

¾              inability to assess the proliferative activity in the dynamics;

¾              inability to detect the proliferative activity in tissues with expected low proliferative activity.

Such methods as enzyme immunoassay, immunohistochemical analysis, immunoblotting, PCR techniques provide high sensitivity, specificity, informativity, but immunohistochemical techniques also provides visibility with the possibility of topical diagnosis. These techniques use monoclonal antibodies produced against specific antigens associated with cell proliferation. For today, there are methods of identifying of such proteins as cyclins, Ki-67, bromodeoxyuridine. We would like to elaborate on some of them.

Proliferating cell nuclear antigen (PCNA) is most commonly identified cyclin; it is an auxiliary protein of DNA polymerase delta. This molecule can be detected in human and animal paraffin-embedded or frozen tissue [Takahashi et al., 1993]. PCNA does not require pre-injection into the tissue under study, as confirmed by the positive results on archival samples, but it has some significant drawbacks:

¾              anti-PCNA immunohistochemistry may give a weak signal in non-proliferating tissues [Takahashi et al., 1993];

¾              the intensity of the signal depends on the method of fixing and amount of pre-heat treatment [Sasaki et al., 1992, Takahashi et al., 1993];

¾              it does not allow evaluating the proliferative activity characteristics and dynamics in in tissues with expected low proliferative activity.

All steps in the application of this methodology held in vitro. The manufacturer recommends a 30-minute incubation period at room temperature. Formalin-fixed and paraffin-embedded tissue sections require unmasking of the antigen with high temperature treatment in10 mMcitrate buffer (pH 6.0) before immunostaining.

Ki-67 antigen, also known as MKI67 - is another endogenous antigen [Scholzen, Gerdes, 2003], a nuclear protein that is bound and may be required for cell proliferation [Bullwinkel et al., 2003]. Furthermore, it is associated with transcription. Inactivation of the Ki-67 antigen leads to inhibition of the synthesis of ribosomal RNA [Bullwinkel et al., 2003, Rahmanzadeh, 2007]. During the interphase Ki-67 can be detected only in the nucleus, whereas in mitosis most of the protein moves to the surface of the chromosomes. Ki-67 is present during all active phases of the cell cycle (G1, S, G2 and mitosis), but it is absent in resting cells (G0) [Scholzen, Gerdes, 2003]. This causes the possible variability of nuclear staining intensity. For detection in paraffin-embedded sections as well as in the case of PCNA thermal pre-treatment is required. Ki-67 can be detected in the tissue without its prior administration. However, it like as PCNA has several drawbacks:

¾              does not assess the features of the proliferative activity in the dynamics;

¾              does not assess the features of the proliferative activity in tissues with expected low proliferative activity.

Cell proliferation index Ki-67 stronger correlated with the index of cell proliferation of bromodeoxyuridine than with PCNA, since the antigen Ki-67 has shorter half-life (1.5 - 2 hours), as compared with PCNA [Bromley et al., 1996], [Bologna-Molina et al., 2013]. All stages of the usage of this methodology held in vitro. The manufacturer recommends a 30-minute incubation period at room temperature or overnight at 4⁰ C. The prior proteolytic processing of formalin-fixed and paraffin-embedded tissue sections is required. The antibodies react with the core antigen of humans and other mammals. Ki-67 protein initially was identified using prototype monoclonal antibodies, which were obtained by immunizing of mice with the nuclei of cells of Hodgkin's lymphoma [Gerdes et al., 1983]. However, currently MIB-1 monoclonal antibodies are exist; they are directed against another epitope of the same antigen. MIB-1 are used in clinical trials to determine the Ki-67 proliferative index. The main advantage of MIB-1 antibodies compared with the original Ki-67 antibodies (and the reason why they substantially replaced the original antibodies in clinical usage) is the possibility of using in fixed paraffin-embedded tissue samples after heat processing, unlike the original antibodies [Bánkfalvi, 2000].

Bromodeoxyuridine (BrdU) is an exogenous proliferation marker; it is synthetic nucleoside analogue of thymidine that is used for studying of the DNA replication [Lehner et al., 2011]. BrdU can be transmitted to daughter cells during the replication [Kee et al., 2002] and defined at least in 2 years after its application [Eriksson et al., 1998]. Enzymes (helicase, topoisomerase) and DNA-binding proteins unwind the DNA and hold the matrix in the untwisted state and rotate the DNA molecule. The correctness of the replication ensures with an exact match of complementary base pairs and DNA polymerase activity, able to detect and correct errors. Method’s principle is based on the detection of BrdU that is capable to replace thymidine during replication, incorporating into new DNA [Lehner et al., 2011]. Anti-BrdU-antibody binds with BrdU, which has been exogenously administered into the newly synthesized DNA, enables the visualization of cells in which DNA replication occurs. The substance can get into the DNA only in S-phase of the cell cycle, thus eliminating the variability of staining [Lehner et al., 2011, Teruaki et al., 2011].

Features of this method are:

¾              it needs pre-injection into the tissue under study, but the exact timing and dosage of BrdU allows for evaluation of the proliferative activity in the dynamics;

¾              prolonged administration of BrdU can be used to assess the proliferative activity in tissues with expected low proliferative activity, since it will be detected in each molecule of the newly synthesized DNA after administration, and transmitted to daughter cells.

BrdU incorporated into the core is highly stable antigen, which gives a strong and reliable signal regardless of the method of fixation and further processing of tissues. Research can be carried out on small rodents. Important qualities of this method are:

¾              low toxicity of the drug (approved for use in humans) [Takao et al., 1985, Takamitsu et al., 2006];

¾              minimally invasive administration, thus reducing the risk of death in experimental animals;

¾              the initial stage of the method is performed in vivo [Lehner et al., 2011].

BrdU does not affect the biochemical constants, neuroautonomic indicators and endocrine balance [Hoshino et al., 1985]. This is an important condition for maintaining of indicators of the simulated pathology. The second investigation phase conducted in vitro on conventionally made tissue sections, which can also be used to perform other histochemical studies. For successful immunohistochemical detection of incorporated bromodeoxyuridine the DNA denaturation is required [Konishi et al., 2011].

For the integrated assessment of the methods above and decision of the most appropriate method for studying of the proliferative activity in the dynamics and in in tissues with expected low proliferative activity we have compiled a table.

Conclusions. Bromodeoxyuridine is a versatile and highly effective marker of DNA replication and cell proliferation, allowing carrying out a dynamic observation of the experiment, including tissues with expected low proliferative activity. It can be widely used in chronic experiments on small rodents, in the simulation of various pathologies and studying the effect of pharmaceuticals on the activity of proliferation.