细胞技术

The functions of the retinoblastoma protein (pRb) are in part regulated by reversible and cell cycle-dependent phosphorylation. While the regulation of pRb by cyclin-dependent kinases (Cdks) has been studied extensively, the role(s) of protein phosphatase 1 (PP1) in controlling pRb a ...

p21WAF1 was originally identified as a protein that binds and inhibits cyclin-dependent kinases (CDKs). p21WAF1 is recognized to have at least two separate roles—first as a CDK inhibitor, and second as an inhibitor of PCNA, an accessory protein of DNA polymerase δ. p21WAF1 plays a critical role in ...

The cyclin-dependent kinase (CDK) inhibitor p21CDKN1A (also known as p21waf1/cip1) is a well known player of the G1 and G2 phase cell cycle checkpoints, which are activated in response to DNA damage. In addition, p21 interacts directly with proliferating cell nuclear antigen (PCNA), thereby ...

In recent years, gene expression quantitation of tumor cells has become of principal importance to analyze gene patterns responsible for cancer development, progression, and resistance to treatment. Whereas semi-quantitative methods, such as Northern blotting analysis, all ...

The activity of cyclin-dependent kinases (CDKs) lies at the heart of cell cycle and checkpoint control. These complexes have been called the “cell cycle engine,” as it is their enzymatic activity that pushes the cells through the cell-division cycle. If this activity is down-regulated, for exa ...

Members of the phosphoinositide-3-kinase-related kinase (PIKK) family, which includes mTOR, ATM, ATR, and hSMG-1, play important roles in regulating the cellular response to environmental stimuli. Despite the similarity of their catalytic domain to that of phosphoinositide-3- ...

Mammalian CHK1 is a Ser/Thr effector kinase that plays critical roles in the DNA damage-activated cell cycle checkpoint signaling pathway downstream of ATR (ATM and Rad3-related protein kinase). This chapter is focused on describing an assay to measure CHK1 activity in vitro. The basic mech ...

Cell cycle checkpoints respond to a wide range of stresses to prevent compromise to the integrity of the cell. The best studied checkpoints are those induced by genotoxic agents that cause DNA damage. Histone deacetylase inhibitors not only increase the acetylation state of chromatin hist ...

It is frequently necessary to examine the biochemical effects of ectopically expressed proteins or short hairpin (sh) RNA-mediated protein knock-down in intact cells. Plasmids that direct the expression of ectopic proteins or shRNAs can be conveniently introduced into cells by tran ...

The G2 checkpoint blocks cells from entering mitosis when DNA is damaged, and helps to protect the integrity of the genome. Tumor cells contain mutations that can inactivate checkpoints, and the inactivation of the G2 checkpoint can induce genomic instability and alter cellular responses ...

Several methods to measure cell cycle perturbation after exposure to ionizing radiation are presented in this chapter. These methods include the G1 checkpoint assay by 5′ bromode-oxyuridine (BrdUrd) labeling followed by flow cytometric analysis, the S-phase checkpoint assay by mea ...

Despite the current availability of an impressive in vitro assay battery developed to quantitatively analyze the broad panel of small compounds and macromolecules that possess the inflammatory potential, little methodology exists nowadays that affords a researcher or clinic ...

Peptide YY (PYY) orchestrates the functions of the gut and the pancreas by regulating growth, digestion and absorption. In addition to its physiological role, PYY exhibits immune and antitrophic properties in the pancreas by decreasing cytokine and amylase release. Although the exact m ...

Differential gene expression represents the entry point for comprehending complex biological processes. In this context, identification and cloning of differentially expressed genes represent critical elements in this process. Many techniques have been developed to fac ...

Cancer has a genomic and proteomic basis. Genomic information provides information about the somatic genetic changes existing in the tumor that provides a survival advantage driving neoplastic progression. On the other hand, proteomics aids in the identification of dysregulated ...

A balance between proliferation and apoptosis is crucial for cellular homeostasis, and its disruption leading to enhanced cellular proliferation and uncontrolled growth are hallmarks of cancer. Genetic manipulation in the mouse offers a powerful approach to delineate the roles of ...

Identification of differentially expressed genes is an essential step in comprehending the molecular basis of complex physiological and pathological processes. Subtraction hybridization and differential RNA display (DDRT-PCR) are two methods that are widely and success ...

An important step in cellular transformation and tumorigenesis is immortalization, in which cells gain the ability to grow indefinitely by bypassing cellular senescence that imposes a finite number of divisions in culture. Primary mouse embryonic fibroblast (MEF) cells have a limi ...

The mammalian innate immune system provides a first line of defense against microbial pathogens and also serves to activate an antigen specific acquired immune program. Key components of innate immunity are the interferons (IFNs), a family of related cytokines with potent antimicrob ...

This chapter will describe methods to assess the activities of protein kinases. Initial studies in the 1950s and 1960s in the field of glucose metabolism examined the activities of several highly specific protein and carbohydrate kinases in cell lysates or isolated cell fractions. As more p ...