Growth Control

Question 1

Cell Division Control

Answer 1

cells in a multicellular organism divide at widely varying rates. Cell division is controlled by internal mechanisms (cell lineage), diffusible substances, cell-extracellular matrix and cell-cell interactions

Question 2

Apoptosis

Answer 2

• programmed cell death occurs during normal development like in formation of the digits- genetic defects lead to syndactyly
• apoptosis also controls the number of neurons during brain development
• it also serves as an important mechanism to dispose of cells after a cell cycle checkpoint error has occurs

Question 3

Trophic factors

Answer 3

• most cells require survival signals to stay alive
• in the absence of these trophic factors (e.g nerve growth factor- NGF) cells activate a “suicide” program
• alternatively, specific signals can induce a “murder” program e.g. in the immune system

Question 4

Morphological characteristics of apoptosis

Answer 4

• cells undergoing apoptosis have distinct characteristics as compared to cells dying as a result of tissue damage or necrosis
• apoptotic cells shrink, and condense and then fragment releasing small membrane-bound apoptotic bodies that are then phagocytosed by macrophages
• the intracellular contents are not released into the extracellular milieu preventing deleterious effects on neighboring cells or inflammation
• in contrast, cells undergoing necrosis swell and burst, releasing their intracellular contents and frequency causing inflammation

Question 5

Signaling mechanisms that regulate the apoptic machinery

Answer 5

• in the absence of trophic factors the pro-apoptotic factor Bad is free to interact with the anti-apoptotic proteins Bcl2 and Bclx inserted in the mitochondrial membrane
• this blocks their inhibitory interaction with Bax permitting the formation of functional Bax-containing ion channels and the release of cytochrome C from the mitochondria into the cytosol
• this results in the activation of a series of cysteine proteases called caspases. Each member is activated via caspase-dependent proteolytic cleavage of an inactive form (procaspase)
• this generates a proteolytic amplification cascade. The caspases digest important intracellular structural proteins such as the nuclear lamins leading to the cells demise and fragmentation
• scientists are attempting to develop therapeutic agents that can selectively target to, and induce apoptosis in tumor cells

Question 6

Terminal Differentiation

Answer 6

-cells stop dividing after a pre-set number of divisions and take on a differentiated phenotype (e.g. neuron, red blood cell

Question 7

Senescence

Answer 7

• cells in culture under ideal conditions stop dividing after 50-100 cell divisions
• since most adult tissue cells lack telomerase, telomeres shorten during each S-phase
• senescence may result from running out of telomeres
• telomerase is a ribozyme, an enzyme that is part protein and part RNA
• it adds a 6-base repeat, GGGTTA, onto the end of the parental DNA strand, to allow lagging strand synthesis to reach the end of the original parental strand
• some cancer cells reactive telomerase, thus avoiding senescence and continuing to divide and form a tumor
• researchers are looking for telomerase inhibitors that may be useful anti-cancer agents

Question 8

p53

Answer 8

• important for blocking entry into S phase following DNA damage or when chromosomes have lost their telomeres
• in this state chromosomes can become unstable resulting in end on fusion and abnormal recombination/breakage events
• replication in this state would be deleterious as growth regulatory genes may be inadvertently duplicated, damaged or lost

Question 9

Growth factors

Answer 9

• diffusible signaling molecules
• effects are concentration and cell-type specific
• growth of cells in culture requires growth factors such as Platelet Derived Growth Factor (PDGF) which are present in blood serum
• some act locally, such as PDGF released from platelets stimulating wound healing
• others act systemically such as erythropoietin (made in kidney) stimulating red blood cell differentiation (in the bone marrow)

Question 10

Cell-substrate (Cell-Extracellular Matrix)

Answer 10

• cells in culture show adhesion-dependence (anchorage-dependent cell growth)
• normal cells fail to divide if they are deprived of interaction with an insoluble matrix i.e. if they are kept in suspension (soft agar)
• this mechanism is used by stratified epithelia to regulate cell proliferation
• in the epidermis of the skin only the cells in direct contact with the basal lamina (the ECM) continue to divide
• cells in the suprabasal layer stop proliferating and differentiate

Question 11

Cell-cell interactions

Answer 11

• normal cells also exhibit density-dependent growth inhibition (cell-cell contact inhibition), i.e. when cells come into contact with neighboring cells they stop dividing
• this phenomenon is observed in vivo during wound repair and can be recapitulated in cell culture

Question 12

Role of cell adhesion to the basal lamina in the maintenance of a tissue organization

Answer 12

• cell type and position is determined at an early stage of embryonic development
• subsequently the cells may proliferate as the embryo grows but the specialized characteristics of a particular cell type remain more of less fixed
• in the adult, cells continue to multiple and die yet the overall tissue organization is maintained

Question 13

Permanent vs regenerative

Answer 13

• some tissues for example most nerve cells and heard cells are permanent; surviving as long as the organism
• other such as liver cells are renewed by simple duplication
• finally, there are tissues that are regenerated from undifferentiated stem cells
• these are required where there is constant need to replace the differentiated cells that cannot themselves divide eg the lining of the gut and the skin epidermis

Question 14

Organization and maintenance of skin tissue

Answer 14

• the overall organization and maintenance of skin tissue requires tightly regulated modification of cell adhesion characteristics as well as changes in gene expression of structural proteins such as intermediate filaments (keratins)
• the tissue, composed primarily of keratinocytes, is organized into several layers
• the undifferentiated stem cells (basal cells) are attached to the basal lamina via members of the integrin family (forming hemi-desmosomes and focal adhesions). This contact critically influences cell fate. Cells expressing the highest number of receptors, and therefore adhering most tightly to the basal lamina are the ones with greater proliferative potential. This phenomenon is known as anchorage dependent cell growth

Question 15

Loss of surface expression of the integrins

Answer 15

• loss of surface expression of the integrins on the stem cells leads to ejection from the basal layer confirming the decision to differentiate and loss of proliferative potential
• as the cells differentiate they express large amounts of intermediate filaments of the keratin family that are involved in the formation of multiple desmosomes between adjacent cells
• this contributes to the strength/barrier characteristics of skin
• ultimately the keratinocytes lose their nuclei and become flattened into squames
• these dead cells are eventually sloughed off to be replaced by the underlying cells
• the time from stem cell division to loss from the skin surface takes 2-4 weeks
• dysregulation of this process contributes to tumor formation and progression

Question 16

Signaling cascades

Answer 16

• proliferation in response to external stimuli involves activation of receptors (like tyrosine) kinases (growth factor receptors) and integrins (adhesion/ECM receptors) and a cascade of second messengers such as the mitogen-activated protein (MAP) kinase cascade
• this leads to increased transcription of early response genes in the nucleus such as myc, fos, and jun
• these are transcription factors that regulate delayed response genes, including cdks and cyclins thereby regulating G1/S cell cycle transition
• normal cell maintain a homeostatic balance of stimulating and inhibiting signals (e.g. kinases vs phosphatases GEFs vs GAPs

Question 17

Oncogenes, Tumor Supressor Genes and Growth Control

Answer 17

• cancer cells do not respond to the normal cell division control signals described above
• they lack: growth factor dependence, anchorage dependences (cells will grow in suspension or in soft agar), cell-cell contact inhibition (cells grow on top of each other forming foci), and do not become senescent
• this is primarily due to altered activity/expression of the proteins that constitute the growth regulatory signaling pathways ie. loss of balance of proto-oncogene and tumor suppressor gene activity

Question 18

Proto-oncogenes

Answer 18

• encode normal cellular proteins that function to stimulate cell growth and division
• these include growth factors, growth factor receptors, tyrosine-specific protein kinases, and transcription-regulating proteins e.g. key intermediates of the MAP kinase pathway
• abnormal regulation of cell growth usually results from either a mutation in the protooncogene leading to a hyperactive or constitutively active product (oncogenes) or from a defect in the regulation of proto-oncogene expression (i.e. more molecules of the normal protein are produced due to gene duplication)
• only one allele of a proto-oncogene needs to be mutated to affect cell growth
• there can be a deletion or point mutation in coding sequence- > hyperactive protein made in normal amounts
• gene amplification- normal protein greatly overproduced
• chromosome rearrangement- you can put it next to enhancer and then the normal protein is overproduced or fusion to actively transcribed gene greatly overproduced fusion protein or fusion protein is hyperactive

Question 19

Examples of Oncogenic Conversion

Answer 19

1) Src non-receptor tyrosine kinase. Mutation of the phosphorylation site involved in the negative regulation of the kinase activity gives rise to a constituitively active kinase

2) The Abl tyrosine kinase. Abnormal translocation of human chromosome 22 and 9 leads to generation of a hybrid gene encoding a Bcr/Abl fusion protein that has constitutive kinase activity
- this is the cause of Chronic Myelogenous leukemia

Question 20

Retroviruses

Answer 20

• many oncogenes were first discovered in Retroviruses
• once again, these are mutated versions of genes (proto-oncogenes) normally present in cellular genomes that were incorporated into the virus genome in the mutated form during its replication cycle in infected cells
• although some animal and plant tumors are caused by viruses (the classical example is the v-src oncogene of Rous sarcoma virus which causes sarcomas in infected chickens) relatively few human cancers (<15%) are virally induced

Question 21

tumor suppressor genes

Answer 21

• tumor suppressor genes such as retinoblastoma (Rb), p53 and DCC (Deleted in Colorectal Carcinoma) encode factors that normally function to inhibit cell growth
• in contract to proto-oncogenes, both alleles of a tumor suppressor must be inactived (by mutation or deletion) before uncontrolled growth occurs. This can often be seen as Loss of Heterozygosity (LOH)
• Rb is an inhibitior of gene transcription. In its active state Rb binds and sequesters the transcription factor E2F. The Rb protein is controlled by a cyclin-dependent kinase (cdk). Phosphorylation of Rb by cdk inactivates it leading to a conformation change and the release of E2F allowing transcription to proceed. Loss of Rb through mutation events leads to constitutive contributing to uncontrolled growth/transformation

Question 22

p53 tumor suppressor protein

Answer 22

• important for checkpoint-control of DNA damage. If DNA is damaged, p53 halts progression through the cell cycle (in part by inducing synthesis of p21, a G1/S cdk inhibitor, which prevents phosphorylation of Rb) and may even induce apoptosis
• if p53 is mutated or missing, even cells with damaged DNA will divide, increasing the probability that new mutations will be seen
• mutations in p53 are very common (50%) in human cancer

Question 23

DNA Viruses and Tumor Suppressor Function

Answer 23

• DNA viruses can interfere with Rb and p53 function by utilizing the host cells to produce proteins normally encoded by the viral genome that bind to and sequester Rb and p53 thereby promoting uncontrolled proliferation and sometimes tumor formation (different from the mutated host genes carried by Retrovirus)
• Papilloma virus (causes Cervical cancer) produces protein E6 and E7 that bind and sequester Rb and p53 respectively
• SV40 virus produces the Large T antigen which binds both Rb and p53 thus these viral proteins can function as oncogenes

Question 24

Stages in Cancer Progression

Answer 24

• most cancers develop through serveral stages, each marked by a mutation in a different proto-oncogene or tumor suppressor gene
• loss of cell division control leads to formation of a tumor or neoplasm, which is benign
• the added ability to invade surrounding tissues (frequency accompanied by loss of the differentiated phenotype) leads to a malignant tumor or cancer
• this is accomplished by a down-regulated of cell-cell adhesion molecules and the production of proteolytic enzymes such as metallo- and serine proteases that degrade extracellular matrix permitting translocation across the basal lamina and into the blood stream
• this can then result in tumor metastasis - the formation of secondary tumors in other organs- frequently the lethal step
• example of the morphologic changes occurring in the epithelium associated with the development of cancer of the cervix is shown below. Anchorage-dependent cell growth regulation (in basal layer) has been lost during the early stages, followed by an invasive phenotype

Question 25

Colo-rectal carinoma

Answer 25

- a total of seven mutations are required for the elimation of function of three tumor suppressor genes, APC, DCC, p53 (two mutations each) and the conversion of one proto-oncogene to an oncogene K-Ras (one mutation) - APC-adenomatous polyposis coli (cytoskeletal/linker protein) - DCC-Deleted in Colorectal Carcinoma (cell adhesion molecule) - Ras- GTPase, component of MAPK pathway - P53- Regulator of DNA repair/cell cycle progression