**2. Cell cycle**

*Cell Growth*

**Figure 1.** *Cell cycle.*

different "nuclear division" processes. Through binary fission, mitosis [8] produces two daughter cells with the same number of chromosomes as the parental cell. Meiosis, also called reductive division, is the division of a germ cell involving two fissions of the nucleus to form four gametes that have half the normal cellular amount of DNA. A male and a female gamete can then combine to produce a zygote, a cell which again has the normal number of chromosomes [9]. Therefore, the enlargement of cell volume and changes in DNA content are two parameters

The cell cycle is tightly regulated at multiple checkpoints [10]. Various growth conditions such as the temperature, nutrients, cell density, and drug treatment can block the cell cycle at various stages. Instead of unrestricted growth, the cell growth curve shows that the cell population increases through three phases: incubation period → exponential hyperplasia → stagnation period. During exponential growth, cells demonstrate great variation in required cell density and doubling

Cell death occurs in each generation. Acute cellular injury causes traumatic cell death (necrosis) [11], whereas apoptosis is a highly regulated and programmed cell death that occurs each day in multicellular organisms. The average adult human loses between 50 and 70 billion cells each day due to apoptosis [12] which is critical, because uncontrolled cell proliferation is closely related to the occurrence of human diseases such as tumors. Commonly used analyses for apoptosis are morphological analysis, detection of apoptotic biomarkers, and flow cytometric analysis of cellular

Inhibiting cancer cell or infectious microbial growth is the purpose of drug treatment. However, drug resistance is the notorious worldwide crisis that prolongs hospital stays and considerably increases mortality. Identification of specific genetic mutations has been the major effort in understanding drug resistance, but the

times that are highly dependent on cell type and growth conditions.

commonly used in cell cycle measurement.

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DNA content.

Each cell cycle involves the G1, S, G2, and M phases in sequence, and each phase is associated with its signature protein biomarkers, DNA content, and cell size, as shown in **Figure 1**. DNA ploidy and protein biomarker analyses are commonly used in cell cycle studies and have important applications in clinical cancer diagnosis, drug efficacy evaluation, prognosis prediction, cell dynamics, and apoptosis.

#### **2.1 Parameters used for DNA content analysis**

DNA ploidy, DNA index (DI), S phase fraction (SPF), and potential doubling time (Tpot) are commonly used parameters for cell cycle analyses.

DNA ploidy refers to the number of chromosomes or the total DNA content in cells. DNA ploidy analysis in combination with clinical pathological diagnosis has a great value in early diagnosis and in prognosis prediction for malignant tumors, for both solid tumors and cancer cell extracts from body fluids, glandular secretions, and exfoliated tissue cells. Aneuploid tumors showed significantly higher recurrent rates than the diploid ones [18].

The DNA index (DI) refers to the ratio between the cells in G0/G1 peak of the tumor samples and that of the normal diploid samples. The calculation equation is DI (DNA index, number of judgment ploidy) = (average number of G0/G1 phase cell peaks in sample)/(average number of G0/G1 phase cell peaks in normal diploid cells). A DI of 1 means a normal diploid sample (generally the normal range is 0.9 to 1.1) [19].

The S phase fraction (SPF) shows the percentage of cells in S phase and indicates the proliferative activity of the cells. The calculation equation is SPF (S phase fraction %) = S cells / (G0/G1 + S + G2M) cells × 100%. Another cell proliferation parameter is proliferation index (PI), and the calculation equation is PI (proliferation index %) = (S + G2M) cells/ (G0/G1 + S + G2M) cells × 100%.

The potential doubling time (Tpot) refers to the time required to double the cell number, which occurs during exponential growth.

### **2.2 DNA content analysis**

DNA content can be analyzed after fluorescent staining or labeled nucleic acid incorporation as shown in **Table 1**.

As shown in **Figure 2**, DAPI and Hoechst dyes penetrate the membrane and are commonly used to label live cells, whereas propidium iodide (PI), propidium monoazide (PMA), and ethidium bromide monoazide (EMA) only label dead cells. Newly synthesized DNA in active proliferating cells can be labeled by the radiolabeled 3 HTdR, 125IUdR [24], or the fluorescent-labeled BrdU and EDU [25].

Flow cytometry measurement (FCM) is a sensitive method to measure cell size and fluorescent labeling. Double staining both cellular DNA and protein biomarker allows identification of cells in G1 peaks, G2+ M peaks, and S platforms, as well as the subdiploid peaks (apoptotic peaks) before the G1 peak. These methods in combination with other biomarkers show the distribution of cells in each phase of the cell cycle and can be used to investigate cell dynamics [26].

### **2.3 Protein biomarkers**

Proteins that are found in proliferating cells, but not in nonproliferating cells can be used as biomarkers for cell cycle measurement. Ki-67 protein (also known as MKI67) is present during all active phases of the cell cycle (G1, S, G2, and mitosis) but is absent in resting (quiescent) cells (G0) [27]. During interphase, the Ki-67 protein is exclusively located in the cell nucleus, whereas in mitosis most of the protein is relocated to the surface of the chromosomes. During cell progression through S phase of the cell cycle, the Ki-67 protein markedly increases [28]. As shown in **Figure 1**, the fluorescent-labeled monoclonal Ki-67 antibody has been used for cell cycle measurement and cancer diagnosis. Other commonly used cell proliferation biomarkers include proliferating cell nuclear antigen PCNA [29], topoisomerase IIB [30], and phosphorylated histone H3 [31].


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*Cell Growth Measurement*

**3. Apoptosis**

**Figure 2.**

protein degradation pathways.

*Methods for DNA analysis in both live and dead cells.*

**3.2 TUNEL method**

small amount of apoptotic cells.

cytometry analysis of cellular DNA content (**Figure 3**).

DNA-specific dyes such as Hoechst dye series and DAPI.

**3.1 Transmission electron microscopic analysis**

*DOI: http://dx.doi.org/10.5772/intechopen.86835*

Apoptosis is a programmed cell death, and the process involves a series of morphological changes such as blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, as well as biochemical changes such as chromosomal DNA fragmentation, global mRNA decay [32], and appearance of protein biomarkers in

Commonly used analyses include transmission electron microscopy for morphological analysis, biochemical assays for detection of apoptotic biomarkers, and flow

Tissue or cells can be directly stained using dyes such as hematoxylin, methyl green pyronine, and acridine orange for microscopic analysis. Under the transmission electron microscope [33], apoptotic cells show reduced size and more concentrated cytoplasm. In the nucleus of pro-apoptosis phase, the chromatin is highly coiled, and many vacuole structures called cavitation appear; in the phase IIa nucleus, the chromatin is highly coagulated and marginalized; in the end, the nucleus is cleaved into fragments and produces apoptotic bodies. Fluorescence microscopy and confocal laser scanning microscopy [34] can also be used to observe the progress of apoptosis, based on morphological changes of nuclear chromatin

The TUNEL assay [35] distinguishes between normal and apoptotic cells based on the amount of FITC-dUTP incorporation into the broken DNA ends in the intact single apoptotic nuclei or apoptotic bodies, whereas the normal or growing cells have almost no DNA breaks and will not be stained. This method accurately reflects the most typical biochemical and morphological features of apoptosis and can used with paraffin-embedded tissue sections, frozen tissue sections, cultured cells, and tissue-separated cells. The method is simple, easy, and sensitive in detecting a very

#### **Table 1.**

*Comparison of methods for DNA labeling.*

**Figure 2.** *Methods for DNA analysis in both live and dead cells.*
