CRITERIA FOR SYSTEMS

• Displays G Protein-Regulated Phenomena• Interactions With Other Signaling Systems• Mammalian Cell• Clonal or Inbred• Nonmalignant/Euploid• Complete Genome Now or Soon• Requisite Mass and Homogeneity• Ability to Modify Gene Expression• Known and Preferably Bidirectional Inputs• Quantifiable and Interesting Outputs• Developmental and/or Pathological Interest• Access to Normal Counterpart

The Cardiac Myocyte

• Regulated Contraction (Gs/Gi-coupled GPCRs) and Secretion in Culture

• Adaptive (Hypertrophic) Responses (Gq-coupled GPCRs)

• Many Cell-Surface Receptors and Signaling Pathways

• Mouse Cardiomyocytes are Problematic• Quantity and Homogeneity of Cells

The Splenic B Lymphocyte

• Homogeneous Populations Isolated Easily (3 Billion/Week)

• Many Cell Surface Receptors and Signaling Pathways• Gi-Coupled GPCRs Mediate Directional Motility in

Response to Chemokines• Mice with Deletions of Genes Encoding B Cell

Signaling Molecules are Often Viable• 1-2 Day Survival in Culture• Availability of Immortal mouse B cell lines

Example – cAMP Double Ligand Screen

AdenylylCyclase

Gs

Adenylyl Cyclase –Centric Model

AdenylylCyclase

Gs

AdenylylCyclase

Gs

Ter LPA

Expression of Membrane-Bound Adenylyl Cyclase Isoforms in

B Cells

AC isoform 1 2 3 4 5 6 7 8 9 Gs

Isoform PCR product AC 1 557 bp AC 2 351 bp*AC 3 691 bp *AC 4 293 bp AC 5 1113 bp*AC 6 649 bp *AC 7 393 bp AC 8 524 bp*AC 9 535 bp

Approaches for Addressing Hypothesis

• Pharmacological Inhibitors

• Knock Out MiceAC1, AC3, AC5, and AC8 have been made

• Antisense or RNAi

•No isoform selective agents are available

•AC3, AC4, AC6, AC7, AC9 expressed in B cells

•Life of B cells in Culture too Short•BCL-2 Mice•WEHI B Cell Line

•Unsuccessful

Experience with AfCS Cell PreparationsProcedure Splenic B Cell WEHI-231 Cardiomyocyte

Prep/Culture ++ Effort Simple ++++ Effort

Longevity Poor, but Bcl-2 Permanent 24 hr, but BDM

Cyclic AMP OK OK OK

Ca2+ OK Ligand set Hard/abnormal

Phosphoproteins OK OK OK

Transcripts Death response ? OK Poor

T’fect/T’duce No Low efficiency Adenovirus

RNAi No Emphatic no ?No

Microscopy No Less than ideal

Lipids OK

Other issues DNA/Prot Cnt’tile proteins

•Is it Time to Reconsider our Choice of cells?

•Are there Better Cells that are More “Experimentally Friendly”?

•What are the Criteria for the Choice of a New Cell Type?

Major Criteria for Cell Selection

1) Possess Interesting (Physiological, Biochemical, or Behavioral) outputs

2) Possess Richness of Inputs (Ligand Responses)

3) Ability to Express (Ectopically) Sufficient Amounts of Proteins for Biochemical Analysis

4) Ability to Alter Gene Expression (RNAi Approaches)

5) Amenable to High Throughput Assays (Intracellular Second Messengers, Phosphoprotein, Microscopy, Transcript Profiling, etc.) Developed by AfCS Laboratories

Testing New Cell Lines

• Exploration of 7 Cell Lines

•Assembled from Suggestions and Input by Steering and Systems Committees, as well as the General

Membership.

•“A” List: J774A.1, Raw 264.7, AtT-20

• “B” List: 3T3-L1, IC-21, Neuro 2a, N1E-115

RAW 264.7•Mouse Alveolar Macrophage Transformed by Abelson Murine

Leukemia Virus•Cells are Capable of Chemotaxis, Pinocytosis, Phagocytosis, and Secretion•Capable of Antibody-Dependent Lysis of Sheep Erythrocytes•Differentiate into Osteoclast-Like Cells•Most Widely Used Macrophage Cell Line (Septic Shock, Lipidomics)•Diversity of Ligand Responses

•Cytokines•Chemokines•Toll-Like Receptors•GPCRs

•Transfection Studies Published•Transcript Profiles Published (LPS)•RNAi reported to work

J774A.1•Macrophage Line Derived from Derived Female BALB/c Mice •Cells are capable of Chemotaxis, Phagocytosis, Secretion (Including Autocrine/Paracrine Factors: NO, TNF- Alpha, IL-6, GM-CSF)•Foam Cell Formation•Respiratory Burst•Growth Inhibition •Diversity of Ligand Responses

•Cytokines•Chemokines•Toll-Like Receptors•GPCRs

•Transfection Studies Published•Infection with Lentivirus•RNAi Reported to Work

AtT-20/ D16v-F2•Mouse, Pituitary Tumor•ACTH Secretion•Epithelial Morphology•Subclone of AtT-20 – Grows as Monolayer•Reported to be Readily Transfectable•Many Ligand Responses (CRH, INE, IL1, IL2, TNF, IL6, INF and , PACAP, VIP, SST, ACh, Activin, ANP/CNP, LIF, Substance P, Glucocorticoids •Antisense Knock down works

IC-21•Murine Line Derived SV40 Transformed Peritoneal macrophage•Cells are Capable of Chemotaxis,Phagocytosis•Less Well Studied than the Other 2 Macrophage Cell Lines•Fewer Reports on Ligand Responses

3T3-L1•Mouse Fibroblast•Well Studied•Transfection by Electroporation and Lipid-Mediated Delivery•Will Differentiate into Adipocyte (IBMX, Insulin, Glucocorticoids), but Preparation is Heterogenous

Neuroblastoma LinesN1E-115

•Mouse Neuroblastoma•So-called “Adrenergic Clone” Derived from C1300 Neuroblastoma (Nirenberg). •Will Differentiate into Neuronal Morphology and “Function”•Ligand Responses (GPCRs Plentiful)•Aneuploid (Modal Chromosome # = 192!!)

Neuro 2A•Mouse Neuroblastoma (Ruddle clone)•Diploid•Less Well Studied Neuroblastoma Line

•Functional outputs: Ionic conductances

Testing New Cell LinesPlan of Attack

• 7 Cell Lines Obtained by Dallas Cell Lab (J774A.1, Raw 264.7, AtT-20, 3T3-L1, IC21, Neuro 2a, N1E-115)

• Leading Candidate (J774A.1, Raw 264.7, AtT-20) Cells are Currently Growing at all Locations (SF,

Stanford, Cal Tech, Dallas)

•Rapid Assessment of “Major Criteria”

Expression of Proteins

•Transfection vs. Infection

•Promoters, Delivery Vehicle, Approaches

•Viral Vectors (Retrovirus, Lentivirus)

•Assess Efficiency (% of cells)

•Assess Expression Levels (amount/cell)

•Is Sorting/Selection Required

Manipulating Gene Expression

Will RNAi Work in these Cells?

Compare siRNA and Pol III Promoter Constructs

Plasmid- vs. Viral-Based Delivery

Richness of Signaling Inputs

•Expression of Receptors

•Literature Search

•Test Ligands for “Commonly Expressed” GPCRs

•Affymetrix Chip Analysis, Most Complete Mouse Chip Available.

Amenable to AfCS Assays

1. Microscopy

a) Morphology

b) Adherence to Desired Substrates

c) Performance of Subcellular Markers

d) Ligand-Induced Domain Translocations (PH)

e) Adequate Transfection Efficiencies and Levels

Amenable to AfCS Assays

2. Phosphoproteins

a) Suitability of Existing Antibody Cocktails

b) Ligand-Induced Changes in Protein Phophorylation

c) Adequate Transfection Efficiencies and Levels for 32P Labeling/Pulldowns

d) Sufficient Material for Pervanadate or Ligand Stimulation/Affinity Purification/Protein ID

Amenable to AfCS Assays

3. Second Messengers

a) Are Ligand-Induced Responses of Sufficient Magnitude?

b) Will Current Methodologies Work?

c) Are Responses Predicted by MA Analysis Present?

Amenable to AfCS Assays

4. Additional Assays

a) Lipid Analysis – Are Cells Appropriate? Do Differentiated 3T3-L1 Present Problems?

b) Protein Traps – is Expression Level of Tagged-Proteins Sufficient?

c) Yeast 2 Hybrid – Additional Prey Libraries?

d) Chemotaxis

e) Phagocytosis, Secretion, Electrophysiology, Others(?)

Is a New Cell Line Better Suited for this Project?

How Quickly Can this be Assessed?

How Much Infrastructure Need be Redeveloped, and How Long Will this Take?

If a Change is Made, When Can We Start the Ligand Screens?