viruses are not the only obligatory intracellular parasite

other unicellular organisms, some bacteial species, some protozoa can multiply only inside other host cells

nucleic acid of viruses

can have EITHER

• DNA
• OR
• RNA

NEVER BOTH!!!

viral reproduction mode

synth. subunits (proteins), then assemble the virus

(very distinct from other things, like bacteria can do fission, etc.)

can viruses grow outside of host cell?

NO

are viruses susceptible to antibiotics?

NO

viruses cannot make ___ independent of host cell

energy or proteins

properties of virus

filterable agents

obligate intracellular parasites

viral components assembled

genome can be DNA OR RNA (never both)

morphology: naked capsid OR envelope

consequences of viral properties

• viruses not living
• must be infectious to endure in nature
• must be able to utilize host cell processes to make their components (mRNA, protein, copies of their genomes, etc.)
• must already have/encode for processes that the cell doesnt provide
• viral components must self assemble

ancient times

• realized that infectious diseases were transmitted by air, water, food, or close contact to sick ppl caused by mysterious elements in fluids called virus
• had ppl w rabies and didnt know what it was
• had ppl w polio and didnt know what it was

effective vaccines against smallpox and rabies developed in 1798 and 1995

no clear understanding of the nature of these disease agents, which are know now as viruses

• didnt understand, just know some things were working

• showed that agent that caused tobacco mosaic disease could passn through fine earth or porcelain filters which retain bacteria
• VIRUSES WERE FIRST DISTINGUISHED FROM OTHER MICROORGANISMS BY FILTRATION

experiments established that certain infectious agents are much smaller than bacteria - called filterable viruses

using filtration as diagnostic tool -> many viruses were discovered in first half of XX century

• 1st tumor virus (RVS, rous sarcoma virus) isolated from sarcomas of chicken in 1911
• years later -> RSV recognized as representative of retrovirus family
• RSV virus which existance of oncogenes was discovered
• wendell stanley found that purified tobacco mosaic virus could form crystals
  • shocked scientific world bc placed viruses at edge btw living organisms and chemical compounds
  • posed question: are viruses living or inanimate???
• CRYSTALLIZATION OF TOBACCO MOSAIC VIRUS CHALLENGED CONVENTIONAL NOTIONS ABOUT GENES AND NATURE OF LIVING ORGANISMS
• Show many properties of life ONLY when inside cell (given means to support replication) because they have ability to mutate, evolve, etc

VIRUSES ARE INANIMATE WHEN THEIR GENOMES PACKAGED IN VIRIONS

• they do share many properties of life:
  • ability to mutate, evolve, reproduce themselves when enter cells that support their replication
• stanley and others showed that viruses contain both protein and nucleic acids
• at the time, it was still not known that genes were made up of nucleic acids
• dev of electron microscope allowed scientists to see viruses for first time (1930s)

possible origins of viruses

see pic

virion components

• nucleic acid - DNA or RNA
• capsid
• nucleocapsid - capsid + nucleic acid core
• envelope
• capsomer

nucleocapsid

capsid + nucleic acid core

naked capsid virus

nucleocapsid

• DNA/RNA
• proteins

enveloped virus

• nucleocapsid
• glycoproteins and membrane

nucleic acid process

• DNA -> RNA -> PROTEIN
• RNA -> DNA in viruses that use reverse transriptase
  • DNA from RNA to make more RNA

viral nucleic acid characteristics

• single vs double stranded
• segmented vs nonsegmented
• linear vs circular
• positive vs negative stranded vs ambisense

enzymes in viral replication

DNA dependent

• DNA polymerase
• RNA polymerase

RNA dependent

• RNA polymerase
• DNA polymerase

viral encoded vs carried in the vial particle

• if virus codes for it, or if it carries it

defective viruses

lack complete genome

= cannot replicate completely/cannot form protein coat

abortive infections

failed infections of a cell

nonpermissive cells wont allow:

replication of a particular type / strain of virus

permissive cells provide:

biosynthetic machinery to support the complete replicative cycle of the virus

viral capsid symmetry

2 types symmetry present in capsids

1. cubic symmetry (icosahedron)
2. helical symmetry
3. neither - ex: poxvirus

cubic symmetry

icosahedron

helical symmetry

• circles inside = nucleic acid
• circles outside = capsomeres that cover and give helical configuration
• for viruses that infect HUMANS
• all these have OUTER ENVELOPE

capsomere is made of what proteins

protomers (= cleaved long proteins made by virus)

they are assembled into pentamers (capsomers)

= how get mature virus form

again, diff btw naked and enveloped virus

naked capsid virus can become enveloped virus (if gains the components)

naked virus = nucleocapsid

enveloped virus = nucleocapsid, AND:

• structural protein = matrix protein
  • only thing inside
• lipid bilayer (outer membrane from host cell)
• glycoprotein - in order to have receptor proteins

virus structure - naked capsid

• only protein structure

naked capsid virus properties

stable to:

• temp
• acid
• proteases
• detergents
• drying

release from cell by lysis

consequenes of naked capsid virus properties

• spread easily (on fomites, hand-hand, droplets, etc.)
• dry out + retain infectivity
• survives gut
• resistant to detergents + bad sewage treatment
• can elicit protective antibody response

naked capsid viruses release from the cell by

lysis

viral structure: enveloped

• proteins
• membrane
• lipids
• glycoproteins

enveloped virus properties

disturbed by:

• acid
• detergent
• drying
• heat

modifies cell membrane during replication

enveloped virus is released from cell by

budding and cell lysis

• bc use membrane of host cell

enveloped virus properties consequences

• must stay wet
• cannot survive GI tract
• spreads in large droplets, secretions, organ/blood transplants
• doesnt need to kill cell (budding)
• initiate cell mediate immune response
• pathogenesis often bc of hypersensitivity and inflammation due to cell mediated immunity

subviral pathogens

• viroids - are only RNA
• prions - are only protein

smaller than viruses

viroids

smallest known pathogens

naked, circular, ssRNA - dont encode protein

only infect plants

comparison of viruses, viroids, prions

baltimore classification of viruses

LECTURE 2

animal cell culture

outline

1. characteristics of animal cell culture
2. growth factors
3. cell cycle
4. cell transformation and elements of cancer

1. characteristics of animal cell culture

animal cells cannot live outside of

their in vivo tissues/environments

a cell culture is the maintenance of cells

in vitro (on glass)

cell culture involves

taking cells from their natural setting, characterizing their growth and functional properties, and keeping them in culture so that they are readily available for experimentation

• start w/ animal cell culture
• culturing is taking cells from vivo environment to conditions in another place they can grow (in vitro)

cells can be grown in culture

cells grown in culture provide more homogenous population of cells from which to extract material, and they are also much more convenient to work with in the lab (in sterile plasticware)

ex vivo cell culture

cells directly from animals/humans

(extracting primary cells from them)

in vitro

In vitro literally translates from Latin as “in glass.” These methods involve experimenting with cells outside a living organism. The original reference to glass is quite literal since in vitro experiments were historically conducted in Petri dishes or test tubes, made of glass.

In vitro cultured cells are purified and isolated from their natural biological environment.

(google)

ex vivo

Ex vivo literally translates from Latin as “out of the living.” In these experiments, living tissues are directly taken from a living organism and immediately studied in a laboratory setting with minimal alterations to the organism’s natural conditions. An example of this is the use of human skin explants derived from surgical procedures.

google

in vivo

these investigations refer to experiments in live animals (NOT in cell cultures)

when conducted in humans = clinical (translational) research

studying animal as a whole

isolating cells and growing them in culture

isolated from INTACT TISSUE where they live in body

• disrupt ECM and cell-cell junctions that hold cells together
  • treat w/ proteolytic enzymes (trypsin, collagenase) = digest + destroy proteins in ECM
  • treat w/ agents as EDTA that bind Calcium ions that cell-cell adhesion depends on
  • tissue can be separated into single cells by agitation

cells can be separated from a mixed cell suspension

• most general cell separation techniques: use antibodies coupled to flourescent dyes/magnetic beads = label specific cells (cell sorting)
• antibody - molecule produced by B lymphocyte, specifically recognizes certain structures of other molecules (antigens)

primary cell cultures

• cell culture established from organs/tissue
• eventually die
  • hayflick limit - limit of division of about 50-70 times
  • experience senescence and death

hayflick limit

• replication capacity of a cell before experiencing senescence and death
• 50-70 divisions
• only occurs in differentiated cells (stem cells can continue to divide)
• this occurs in our bodies as well

senescence due to

progressive shortening of telomeres (with each cell division)

telomeres

repetitive DNA caps at end of each chromosome

telomerase

elongates telomeres, which are shortened after each cell division

cells stop making telomerase, which is why their telomeres shorten with each division -> leading to eventual senescence and death of the cell

cancer cells express

telomerase continuously

since they express it, their telomeres do not shorten, and thus, cancer cells are immortal

fate of primary cells

senescence

• cells stop proliferation at hayflick limit after certain number divisions
• if they remain normal cells, senescence will occur
• cell strain
• euploid
• before senescence is reached, cells may be changed by viruses, carcinogens, etc or spontaneous transformation -> cell line

cell strain

euploid (normal # chromosomes) population of cells

immortalization

• cell line
• aneuploid (abnormal # chromosomes or changed chromosomes)
• occurs if primary cells (normal cells) undergo transformation into abnormal ones
• can be cultured indefinitely due to telomerase continuation

cell line

aneuploid (abnormal # or changed chromosomes)

can culture indefinitely bc of telomerase activity continuation, do not die

normal cells can turn into abnormal cells (cell line) by carcinogens, transforming viruses, spontaneous transformation, etc.

transformed cell line

• line of cells derived from the cell lines
• in vitro characteristics of tumor cells (foci)
• can turn into these bc of carcinogens, etc. (stated before)

tumor cell lines

cell line derived from primary tumor cells (ex vivo tumor cells, from an organism)

transformed cell lines and tumor cell lines grow...

without attaching to a surface and proliferates to much higher density in a culture dish

normal cells treated w/ carcinogenic chem compounds or with transforming viruses can result in...

transformed cell line

transformed cells injected in mice can cause

tumors in mice

if smth injected in mice and cause tumors, the chemical is considered

carcinogenic

short and long term test for genotoxicity and carcinogenicity (transformation test)

cell lines differ in important ways from normal progenitors in tissues from which they were derived

see pic

cell culture medium

• must resemble blood plasma, so has things to make it as similar as possible
  • amino acids, vitamins, glucose, buffers, serum, antibiotics, etc. additives

types of cells in cell culture

all animal cells in culture derived from animal tissue

• adherent vs suspension

cell types

1. primary cells - eventually stop replicating
2. continuous cell lines
   • transformed/tumor/spont. altered cells
   • stem cells

stem cell line

stem cells (self renewing) cultured in vitro and can be propagated indefinitely

derived from either animal or human tissues and come from 1 of 3 sources

• embryonic stem cells
• adult stem cells
• induced stem cells

properties of primary cells

• euploid
• high serum conc.
• finite lifespan/hayflick limit
• display properties of differentiated cells
• respond to modulators of cell growth (growth factor, inhibitor, etc)
• do NOT produce tumors
• adherent cells - need contact with solid surface for division (anchorage dependent growth)
• subject to contact inhibition - stop growing when touch neighbor cell, grow again when have space

properties of continuous cells (cell lines)

• aneuploid
• may have anchorage independent growth
  • dont need to be anchored to duplicate
• immortal - divide forever bc telomerase
• do not respond to neighboring cells - no contact inhibition
• dont display properties of differentiated cells
• do not respond to modulators of cell growth

effect of virus infected cells

exert modifications on cells

• cytopathic (cytolytic) viruses
  • kill their host cells (lysis)
  • either completely eliminated by immune system or kill infected organism (lysis)
• noncytopathic viruses (LCMV)
  • propagate without killing their host cells
  • leave cell via other mechanisms
• persistent virus
  • ex: herpes virus, chickenpox, shingles
  • lytic/latent life cycle
  • CANNOT be cured of this virus
    • virus hides until immunosuppression, then comes out
  • cytopathic or cytopathogenic effects (changes in host cell caused by viral invasion)
    • change morphology
    • apoptosis
    • necrosis
    • cell fusion (syncytia)
    • hemagglutination
    • change in growth or lifespan
    • oncogenic transformation

cytopathic (cytolytic) viruses

• kill their host cells (lysis)
• either completely eliminated by immune system or kill infected organism (lysis)

noncytopathic viruses (LCMV)

• propagate without killing their host cells
• leave cell via other mechanisms

persistent virus

• ex: herpes virus, chickenpox, shingles
• lytic/latent life cycle
• CANNOT be cured of this virus
  • virus hides until immunosuppression, then comes out

apoptosis

active, programmed process of autonomous cellular dismantling occurring in a cell, that avoids eliciting inflammation

necrosis

passive, accidental cell death of a group of cells resulting from environmental pertubations with uncontrolled release of inflammatory cellular contents

some viruses use necrosis, some use apoptosis

cell fusion (syncytium)

viral proteins that mediates fusion of an infected cell with neighboring cell

• lead to formation of multinucleate cells - syncytia

virus w/ this puts molecules on surface that cause fusion of cell w/ neighboring cell

ex: flu, COVID, syncytium resp. virus

hemagglutination

rxn that causes clumping of RBC in presence of some enveloped virus

glycoprotein on viral surface (hemagglutinin) interacts w/ RBCs leading to clumping of RBCs

virus attach to RBC and agglutinate

ex: flu

RSV causes

tumors

is a transforming virus

primary cells / cell lines / immortality/ telomers / telomerase: summary

which are cells that can replicate?

• in vivo - in embryo and adults
  • embryo - embryonal cells, stem cells
    • pluripotent - undifferentiated, can become any cell type
  • adult - adult stem cells
    • multipotent - restricted to become any type of cell in the tissue/organ they reside (bone marrow, fat, brain, blood vessels, skin, teeth, heart, liver)
    • adult stem cells replace cells lost in tissue as needed (ex: skin growth every day)
  • benign + malignant (cancer) tumor cells
• telomerase expressed/activate in tightly controlled + regulated fashion, except in malignant cells where it is cont. expressed/activated and they are then immortal

Embryo stem cells - Pluripotent – undifferentiated, can become any type of cell

Adult stem cells – multipotent

- replace cells lost in tissue/organ they reside as needed

Benign and malignant tumor cells

- tumor cells can be benign – have capacity to replicate, but do not metastasize and invade

- telomerase expressed regulated/controlled

which are cells that can replicate?

now in vitro:

• primary cells - limited capacity: hayflick limit, telomers shorten progressively, senescence, death
• telomerase continuously expressed/activated enabling cell immortality in the following:
  • spontaneous generated cell lines
  • transformed cell lines
  • tumor (cancer) cell lines

telomerase - (an enzyme) is a ribonucleoprotein complex

• catalytic core: telomerase reverse transcriptase (TERT)
• noncoding human telomerase RNA (hTR) which serves as template for the addition of telomeric repeats to chromosome ends

cellular senescence is a permanent proliferation arrest that occurs in response to:

endogenous and exogenous stresses, including telomere dysfunction

also to oncogene activation, tumor suppressor genes inactivation, oxidative stress, mitochondrial dysfunction, and persistent DNA damage

senescence occurs at the ___ levels and is related to the ___ process but not necessarily to death

• cellular, tissue, organ, and body levels
• related to the aging process

senescent cells start producing what molecules?

inflammatory

II. GROWTH FACTORS

part 2 of lecture

• secreted proteins which exert their effects at very low concentrations (1-^-9 to 10^-11 M)
• regulate protein synthesis and cell growth
• act on cells that express specific receptors
• can stimulate or inhibit proliferation or differentiation
• over 60 known growth factors
  • broad specificity
  • narrow specificity

growth factors = proteins

growth factors and their actions

the main growth factors

dont need to memorize

produced by diff cell types

macrophages make most types of growth factors

signaling pathways

Growth factors act on specific receptors

- end: give info in nucleus for cell to express certain thing

Interact w/ receptor -> phosphorylate -> kinases activated/phosphorylated

• Kinases go to nucleus -> activate gene/regulatory proteins
• Genes have to do w/ expression, regulation etc.

4 types chem signaling

• autocrine
• across gap junctions
• paracrine
• endocrine

autocrine

• cell targets itself
• signaling acts on the signaling cell
• 1 cell produce growth factor that goes back to interact w/ growth factor on same cell that produced it
• Bc only way to induce this function, must go out and activate the cell signaling pathway

signaling across gap junctions

• cell targets a cell connected by gap junctions
• signaling molecules moving directly between adjacent cells
• internal receptors found in cell cytoplasm
• bind ligand molecules that cross the plasma membrane

paracrine

• a cell targets a nearby cell
• signaling act on nearby cells
• Growth factor act on receptor in neighbor cell

endocrine

• a cell targets a distant cell through the bloodstream
• signaling uses the circulatory system transport ligands

III. the cell cycle

part 3

1. the cell cycle, or cell division cycle, is the series of events that take place in a cell leading to its division and duplication (replication)
2. cell cycle can be divided in 2 periods
• interphase
• cell growth
• mitosis
• cell division into 2 daughter cells
3. cell cycle consists of four distinct phases
• interphase

1. G1 phase
2. S phase
3. G2 phase

• 4. M (mitosis)

cell cycle can be divided in 2 periods

• interphase
• cell growth
• mitosis
• cell division into 2 daughter cells

cell cycle consists of four distinct phases

• interphase

1. G1 phase
2. S phase
3. G2 phase

• 4. M (mitosis)

cell cycle checkpoint

• checkpoints used by cell to monitor + regulate progress of the cell cycle
• checkpoints
• G1 - ensure ready for DNA synthesis
• G2 checkpoint - ensure ready to enter M phase and divide
• if not, mitosis avoided
• metaphase checkpoint - ensure cell ready to complete cell division
• chromosomes aligned?

cells can enter nongrowing G0 state

due to serum deprivation of the proliferating cell in culture -> arrest and into G0 state

protein synth. rate decreases to 20%

cannot pass G1 checkpoint/go into G1

cell cycle synchronization

• process by which cells in a culture at diff stages of cell cycle are brought to the same phase
• vital for cell study -> population-wide data

2 types of cell synchronization

1. physical fractionalization
2. chemical blockade

can do synchronization by

1. serum starvation
2. chemically
   • nocodazole - arrest at metaphase
   • hydroxyurea - block in S phase
   • mimosine - late G1 arrest

IV. cell transformation + elements of cancer

Generation of transformed cells (mechanisms of cell transformation)

• culturing of primary cells for long periods (rodent cells)
• mutagenesis (genotoxic chemical compounds or other means)
• mutagens can transform the cells
• tumor viruses (HTLV-1)
• viruses can transform the cells
• transfection with oncogenes
• oncogenes can be responsible for inducing cancer
• can be isolated from tumors (= tumor cells)

malignant cell transformation refers to "initiation" first step on carcinogenesis model

• as occurring in any cell w/ initiating first mutation in any gene relevant to cancer
• malignant cell transformation can be achieved by tumor viruses, chemical carcinogens, etc.

- initiation - initiated cell

-> cell proliferation

-> promotion (altered cell foci prenoplastic lesion)

-> genomic instability and increased cell proliferation (addl genotoxic and promoting carcinogens)
-> progression (invasion and metastasis) = cancer

= carcinogenesis somatic mutation theory model

Model of cancer genesis

Malignant transformation – initiation – when cell turns cancer

Cancer progress through years from 1st cell’s initiation

Initiated cell can be repaired by DNA repair mechanism

- if DNA mutation remains, then another body system finds the cell and eliminates it

- if still have tumor cell, then these two systems failed to eliminate it

- then cell will proliferate -> become tumor

- progression – when benign tumor has a cell that then invades blood vessel and metastasizes

initiation step of malignant cell transformation

• as occurring in any cell w/ initiating first mutation in any gene relevant to cancer
• malignant cell transformation can be achieved by tumor viruses, chemical carcinogens, etc.

qualities of transformed cells

• immortalization
• aberrant growth control
• loss of contact inhibition
• anchorage independence (colony formation in soft agar)
• tumorigenicity (tumor formation in animals)
• malignancy (formation of an invasive tumor in vivo)

note: transformation is a multistep process, and varying degrees of transformation are measurable

molecular determinants in the conversion from normal to the malignant cellular phenotype: "hallmarks of cancer"

• growth signals
• self-sufficiency in growth signals
• dont need info from growth factors
• insensitivity to anti-growth signals
• cell division
• limitless potential for cellular replication
• escape from apoptosis
• oncogenesis
• tissue invasion and metastasis

cancer cannot be described in vitro, but only in vivo (invasion and metastasis)

Diagnosis in cancer can ONLY be done by a biopsy – shows invasion + metastasis

- invasion when a cell of the benign tumor breaks the yellow membrane and goes into extracellular matrix and into blood

what classifies as cancer?

cancer cannot be described in vitro, but only in vivo

invasion and metastasis

Diagnosis in cancer can ONLY be done by a biopsy – shows invasion + metastasis

- invasion when a cell of the benign tumor breaks the yellow membrane and goes into extracellular matrix and into blood

2 classes of genes in cancer

• oncogenes
• tumor suppressor genes

proto-oncogenes

NORMAL genes in a genome

normal cells that stimulate cell growth and division

Proto-oncogenes are genes that normally help cells grow and divide to make new cells, or to help cells stay alive.

ex: growth factors, mitogens, receptor tyrosine kinases, serine-theronine kinases, GTPases, transc. factors, etc.

oncogene

When a proto-oncogene mutates (changes) or there are too many copies of it, it can become turned on (activated) when it is not supposed to be, at which point it's now called an oncogene.

cause cancer thru gain of function

one of main characteristics of cancer - uncontrolled growth

causes transformation of normal cells into tumor cells

can activate by diff mechanisms (mutagenesis, amplification, mutations, chromosomal translocations, etc.)

ex: Ras, HER2, Myc, Cyclin D

activated proto oncogene

oncogene

gained function

more about proto-oncogenes

• important regulators of biologic processes
• do NOT reside in genome for purpose of promoting tumors
• they are normal biologic process
• play role in cell growth (proliferation, apoptosis, genome stability, differentiation, etc.)
• causes transformation of normal cells into tumor cells

oncogenes associated with retroviruses

(see pic)

ex:

• tyrosine -> abl oncogene -> cause sarcoma in cat
• serine/threonine -> raf oncogene -> cause sarcoma in chicken/mouse
• etc.

cellular oncogenes and their functions

diff functions of growth factors

• all are proto-oncogenic functions required by healthy cell to function
• if any activated -> then have an oncogene

tumor suppressor genes

• provide negative control of cell proliferation in normal genes
• loss of function of proteins encoded by these genes liberated cell from growth constraints -> contributes to malignant control
• are BRAKES in proliferation of cells
• both copies of tumor suppressor gene must be mutated/lost to result in loss of growth control
• they are recessive genes
• ex: Rb, p53, BRCA1/2

mechanisms of tumor suppressor gene inactivation

• deletion
• point mutation
• mutation followed by duplication
• loss of heterozygosity (LOH)
• DNA methylation
• post-transc. mech-bindind to DNA viral oncoproteins
• epigenetic silencing

oncogenes + tumor suppressors

• oncogenes - accelerators
  • 1 mutated is enough for excessive cell proliferation
• tumor suppressor - brakes
  • BOTH genes must be inactivated to yield excessive cell proliferation

viruses associated w/ human cancer

HPV

(DNA)

cervical carcinoma

Hep B

(DNA)

liver cancer

herpesvirus: epstein barr virus

(DNA)

burkitt's lymphoma

herpesvirus: HHV8

(DNA)

kaposi's sarcoma

Hep C
(RNA)

liver cancer

retrovirus family

(RNA)

adult T cell leukemia