Biology of Cultured Cells

Transcription

1 Biology of Cultured Cells

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3 Transfection of cells (particularly human cells) with RAS or RAF genes

4 Transformation transfection or DNA transfer Transformation of cultured cells implies a spontaneous or induced permanent phenotypic change resulting from a heritable change in DNA and gene expression. Transformation is associated with genetic instability and three major classes of phenotypic change: Immortalization. aberrant growth control, the loss of contact inhibition of cell motility, density limitation of cell proliferation, and anchorage dependence. malignancy, as evidenced by the growth of invasive tumors in vivo

5 Immortalization The acquisition of an infinite life span Immortalization of murine cells requires circumventing at least p19arfp53. In human cells must be treated with exogenous agents (e.g. ionizing radiation, chemical mutagens/carcinogens, viruses) to induce this change. However, in the absence of telomerase expression the cells telomeres become extremely short, and the cells enter a period of proliferative arrest ( crisis ) often accompanied by abnormal morphology and degenerative loss in cell number.

6 Cancer cells can escape crisis by expressing telomerase Telomere collapse and crisis would appear to be irreversible processes from which cells can never escape. Telomere regeneration can be accomplished through the actions of the telomerase enzyme, which functions specifically to elongate telomeric DNA. Telomerase activity is clearly detectable in 85 to 90% of human tumor cell samples. In normal cells telomerase activity is very minor except of the germ cells of the testes, and lymphocytes. Furthermore, expression of the telomerase enzyme is present early in embryogenesis and is largely lost during the cellular differentiation.

7 Telomerase Although most of the normal human cells have the gene for this enzyme but its not expressed. Structurally, at the core of the mammalian telomerase holoenzyme are two subunits. In human cells, this catalytic subunit, termed htert (for human telomerase reverse transcriptase) it is a DNA polymerase using its own RNA template the second essential subunit. It synthesizes a DNA molecule that is complementary to six nucleotides present in the telomerase-associated RNA molecule

8 htert uses htr as a template The holoenzyme attaches to the 3ʹ end of the G-rich strand overhang through the hydrogen bonding of htr to the last five nucleotides of the G-rich strand. Subsequently, by reverse transcription of sequences in the htr subunit, htert is able to extend the G-rich strand by six nucleotides. Upon completion of this elongation, a conventional DNA polymerase can fil in the complementary (blue) strand.

9 Immortalization Should telomerase expression then occur spontaneously or be induced, this can lead to immortalization Mouse fibroblasts, appear to have natural telomerase expression. In vitro transformation is not equivalent to neoplastic transformation, which is the ability of cells to form tumours when injected into an immunoincompetent host. However, the belief appears to be emerging that cells that are both transformed and immortal are probably potentially tumorigenic.

10 Terms Primary culture? Subculture (also called passage)? Cell line? Cell strain? Clonal culture (clone)?

11 The characteristics of normal and transformed cells Normal cell: Anchorage dependence: Monolayer culture:

12 Media composition, ph, osmolality and the volume and frequency of replenishment. Incubation conditions such as temperature, relative humidity and gas composition can be regulated, as can the form and composition of the physical substrate for cell attachment

13 The Culture Environment Often the cell does not behave as in vivo because: 1- loss of heterogeneity and three-dimensional architecture. 2- Hormonal and nutritional stimuli are not the same! Cells commonly favor the spreading, migration, and proliferation of unspecialized cells, rather than the expression of differentiated functions. How does environment affect culture? Nature of the substrate Degree of contact with other cells Constitution of the medium Constitution of the gas phase The incubation temperature

14 Cell Adhesion Solid tissues grow as adherent monolayer, and unless they have transformed and become anchorage independent What is required for proliferation? - Glass with a slight net negative charge and some plastics, such as polystyrene treated with strong acid, a plasma discharge, or highenergy ionizing radiation - Spreading may be preceded by the cells secretion of extra cellular matrix proteins and proteoglycans. - To bind to the matrix via specific receptors

15 Cell Adhesion 2 nd hand glass or plastic, are they Ok to use? Fibronectin or collagen, or derivatives such as gelatin can also help cells to and Epithelial cells require cell cell adhesion for optimum survival and growth, and consequently they tend to grow in patches. while fibroplasts have other features.

16 What cells grow in colonies and what don t care? FG0 : Skin fibroblast MCF7 : Breast adenocarcinoma

17 Cell Adhesion Molecules To understand this you need a short revision about tissues in vivo. CAMs and cadherins are depicted between like cells, and integrins and proteoglycans between the epithelial layer and the matrix of the basal lamina.

18 Connective tissue Epithelial tissue Muscles Nerves Tissues In connective tissues, extracellular matrix is plentiful and carries the mechanical load. ( The ECM is composed of a large collection of biochemically distinct components including proteins, glycoproteins, proteoglycans, and polysaccharides with different physical and biochemical properties) We discuss connective tissues first

19 connective tissues Many types of CT. In all of these tissues, the tensile strength whether great or small is chiefly provided by a fibrous protein: collagen. The various types of connective tissues owe their specific characters to the type of collagen that they contain, to its quantity, and, most importantly, to the other molecules that are interweave with it in varying proportions (elastin, as well as a host of specialized polysaccharide molecules)

20 Collagen Mammals have about 20 different collagen genes. They constitute 25% of the total protein mass in a mammal long, stiff, triple-stranded helical structure, in which three collagen polypeptide chains are wound around one another in a ropelike super helix. The cell in the photograph is a fibroblast, which secretes the collagen as well as other extracellular matrix components Other collagen molecules decorate the surface of collagen fibrils and link the fibrils to one another and to other components in the extracellular matrix

21 Collagen In skin, tendon, and many other connective tissues they are called firoblasts. The cells secret collagen molecules in a precursor form, called procollagen, with additional peptides at each end that obstruct assembly into collagen fibrils. Extracellular procollagen proteinases cut off these terminal domains to allow assembly only after the molecules have emerged into the extracellular space.

22 Collagen Cells in tissues have to be able to degrade matrix as well as make it. Matrix proteases ( arthritis and cancer) cells organize the collagen that they secrete Integrins couple the matrix outside a cell to the cytoskeleton inside it

23 Collagen-fibronectin Cells do not attach well to bare collagen. Another extracellular matrix protein, fibronectin, provides a linkage: one part of the fibronectin molecule binds to collagen, while another part forms an attachment site for a cell. Cell attaches itself to this specific site in fibronectin by means of a receptor protein, called an integrin, which spans the cell s plasma membrane.

24 Integrins Integrins do more than passively transmit stress: They also react to stress and to chemical signals from inside and outside the cell that direct them to maintain their attachment to other molecules or to let go. Integrins form and break attachments, for example, as a cell crawls through a tissue, grabbing hold of the matrix at its front end and releasing its grip at the rear.

25 Integrins perform functions by undergoing remarkable conformational changes

26 Integrins Binding to a molecule on one side of the membrane causes the integrin molecule to stretch out into an extended, activated state so that it can then latch onto another molecule on the opposite side an effect that operates in both directions across the membrane In this way, the external attachments that a cell makes help to regulate whether it lives or dies, and if it does survive whether it grows, divides, or differentiates. Humans make at least 24 different kinds of integrins leucocyte adhesion deficiency

27 Integrin and cell survival Under normal conditions, when integrins on the epithelial cell surface come in contact with the ECM, FAK is activated by phosphorylation, which in turn triggers a phosphorylation cascade ending with the activation of Akt, thus promoting cell survival. If the integrin should lose contact with the ECM, the cell survival signals cease, leaving pro-apoptotic proteins such as Bad uninhibited and able to initiate cell death. Cancer cells can overcome anoikis in a variety of ways that are often related to EMT

28 What cancer does to migrate In order to migrate, cancer cells must activate genes for slow down proliferation events,, activate anti-apoptotic mechanisms ( loss of E-cadherin expression and an increase in N-cadherin expression is correlated with anoikis resistance and increased invasiveness). Alter cellular characteristics from epithelial to mesenchymal, down- regulate the receptors that aid in cell-to cell attachment. up-regulate the cell adhesion molecules that help in cell movement, degrade cell-to-cell junctions, activate proteases at the cell surface in order to cut through the extracellular matrix

29 a mesenchymal phenotype, is judged by loss of epithelial cadherin (E-cadherin) expression and the expression of vimentin and neural cadherin (N-cadherin)

30 Integrins in cancer: biological implications and therapeutic opportunities The integrin family of cell adhesion receptors regulates a diverse array of cellular functions crucial to the initiation, progression and metastasis of solid tumours. The importance of integrins in several cell types that affect tumour progression has made them an appealing target for cancer therapy. Integrin antagonists, including the αvβ3 and αvβ5 inhibitor cilengitide, have shown encouraging activity in Phase II clinical trials and cilengitide is currently being tested in a Phase III trial in patients with glioblastoma.

31 What is else? Gels of polysaccharide and protein Fill spaces and resist compression Complementary function for proteoglycans, extracellular proteins linked to a special class of complex negatively charged polysaccharides, the glycosaminoglycans (GAGs)

32 Proteoglycans Proteoglycans are extremely diverse in size, shape, and chemistry. Typically, many GAG chains are attached to a single core protein, which may in turn be linked at one end to another GAG, creating an enormous macromolecule resembling a bottlebrush, with a molecular weight in the millions of daltons. Hyaluronan A glycosaminoglycan defined by the disaccharide unit that is neither sulfated nor covalently linked to protein. It is referred to in older literature as hyaluronic acid.

33 It is not the same for all CT! Bone have collagen plus calcium phosphate crystals. jellylike substance in the interior of the eye consists almost entirely of one particular type of GAG, plus water, with only a small amount of collagen GAGs have multiple negative charges attracting a cloud of cations, such as Na+, that are osmotically active, causing large amounts of water to be sucked into the matrix In general, GAGs are strongly hydrophilic and tend to adopt highly extended conformations, which occupy a huge volume relative to their mass.

34 Proteoglycans, can bind secreted growth factors and other proteins that serve as signals for cells They can block, encourage, or guide cell migration through the matrix

35 Focal Adhesions Anchoring Cells to Their Substratum At first, the cell has a rounded morphology it sends out projections that form increasingly stable attachments

36 Focal adhesions When fibroblasts or epithelial cells spread onto the bottom of a culture dish, the lower surface of the cell is not pressed uniformly against the substratum. Instead, the cell is anchored to the surface of the dish only at scattered, discrete sites, called focal adhesions. Focal adhesions are dynamic structures that can be rapidly disassembled if the adherent cell is stimulated to move or enter mitosis.

37 Focal adhesions are sites where cells adhere to their substratum and send signals to the cell interior Transmembrane proteoglycans also interact with matrix constituents such as other proteoglycans or collagen act as lowaffinity growth factor receptors.

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