Chapter 1. To Kill A Cancer Cell

1.1 Introduction

Lodish 8e Case Studies

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To Kill A Cancer Cell

Signal Transduction Case Study

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Instructor's Notes

This case study investigates the process of cell signaling and how signaling pathways can be modified in cancer cells. The student will become well versed with several different methodologies, and will use this knowledge in both data interpretation, as well as experimental design.

These protocols include:
• PCR
• RT-PCR
• Western blotting,
• Immunoprecipitation
• Immunofluorescence microscopy

In order to successfully complete this simulation, students must understand four basic cell-signaling pathways from ligand-receptor interaction to activation of transcription factors. These four pathways are:
1. Tyrosine kinase receptors (Section 16.3, p. 734)
2. G-protein coupled receptors (Section 15.3, p. 686)
3. Serine receptor kinases (Section 16.1, p. 722)
4. Wnt signaling (Section 16.6, p. 751)

Specifically, there is an emphasis on:
• Receptor activation, including homodimerization and heterodimerization
• Ligand-specific interaction with receptors
• G-protein activation
• Stimulation of downstream signaling molecules
• Activation of transcription factors and their subsequent change in subcellular localization

Students are awarded points for answering questions throughout the activity, for correctly identifying key components in each signaling pathway, interpreting the data, suggesting follow-up experiments, and so forth. Each question is worth 1 point. Multiple choice questions are automatically graded; students are given one opportunity to correct an incorrect answer before moving on. Short-answer questions are accepted provisionally; instructors must evaluate the answers and determine whether they are acceptable. The final score is recorded in the gradebook.

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Figure 1.1

As the lead scientist at the largest pharmaceutical company in North America, PharmUSA, Kaitlin Summers is in charge of developing new drugs to combat cancer. It has been a long year, but her team has developed several new drugs for preclinical testing in cell lines, and even more are in the pipeline. Because she frequently partners with colleagues in academia on projects, she decides to give one of her old lab mates, Rick Stanton, a call. She and Rick got PhDs in the same lab at Cal State.

After spending a few minutes catching up on their personal lives, they start talking science. Rick remarks, “We've generated some data from pancreatic tumor samples that we've been collecting over the past year."

He continues, “We've got nearly 100 samples and have generated cell lines from most of them. Almost 80 percent of these cancer cell lines are secreting an uncharacterized protein we're calling 'protein X.' It's a 90-kDa protein that appears to stimulate cell proliferation and survival. The pancreatic cancer cell lines expressing this protein are from those cancers that have shown metastases, so they are more aggressive than the cell lines that don't express it. If we can figure out the signaling pathway that's responsible for its expression … "

Kaitlin finishes his sentence “ … then we can target that pathway with an inhibitor drug and kill the cancer cells."

Rick agrees to send 10 pancreatic cancer cell lines that express protein X, 2 pancreatic cancer cell lines that do not express protein X, and a cell line derived from normal pancreatic tissue.

After getting off the phone, Kaitlin sets up a conference call with the research heads of four divisions that are looking at different signaling pathways, and they collectively agree to convene the following day to discuss the project.

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Question 1.1

Eub2M9okGdggWdfIuQb2DzLuWORiqUmBypIUlUxq9/z0BQWZ4xbUOLlfiTh9STDS2w7JjAEAGoZxrKoc2y4EbA==

The question Kaitlin's research group is trying to answer is: Which signaling pathway activates expression of protein X?

The next day, Kaitlin’s research group convenes. Amber Collins leads the receptor tyrosine kinase group. Tom Welch is head of the G protein–coupled receptor group. Dan Tucker is in charge of the TGF-β signaling group. Sarah Wentworth leads the Wnt signaling team.

After the group discusses Rick’s preliminary findings with the pancreatic cancer cell lines, they decide that the best approach is to identify the signaling pathway responsible for the expression of protein X. Once that pathway is determined, the drug development team can test pathway inhibitors to selectively kill the cancer cells. Kaitlin wishes the team good luck before they adjourn.

Each of the labs is set up for the following protocols:

PCR (polymerase chain reaction): A technique for amplifying a specific DNA segment in a complex mixture by means of multiple cycles of DNA synthesis from short oligonucleotide primers followed by brief heat treatment to separate the complementary strands (see Figure 6-18)
RT-PCR: Amplification of a specific mRNA in a complex mixture using a reverse transcriptase step followed by PCR (see Figure 6-19)
Western blotting: Identification of a specific protein in a complex mixture by means of gel electrophoresis and specific antibodies (see Figure 3-41)
Immunoprecipitation: A technique that uses antibodies to separate a target molecule of interest from other molecules in a complex mixture in solution by cross-linking the target molecule into a large aggregate, resulting in the formation of an insoluble solid (precipitate) that can be easily separated and analyzed (see Figure 3-41)
Immunofluorescence microscopy: A technique used to localize a specific protein in a cell using an antibody with a fluorescent tag (see page 144, Figure 4-14)

1.2 Amber's lab: Receptor tyrosine kinase pathways

Amber's lab: Receptor tyrosine kinase pathways

After a few days, Kaitlin stops by Amber's lab to check in. Amber gives the following report:

"Good morning! During our analyses of these cells, we've found identical results in all 10 of the aggressive pancreatic cancer cell lines expressing protein X. If we grow the cells to the point that they form a monolayer that covers the plate, they really start to express protein X more robustly. We've had to keep them at about 50 percent coverage to dissect out contributions from different signaling pathways. The 2 nonaggressive pancreatic cancer cell lines have also shown identical results. Dan, Tom, and Sarah have found the same to be true in their experiments. To simplify the data analysis, it's easier to look at the results from each research group. Let's look at what we've found."

true

1. PCR

"We did not do PCR, but we did do RT-PCR assays."

true

2. RT-PCR

"We isolated RNA from the cells, made cDNA using a reverse transcriptase protocol, and then used primers specific for HERs (human epidermal growth factor receptor tyrosine kinases) in an RT-PCR assay. Here are the data."

RT-PCR results show the following lanes and bands: Normal pancreatic cells and RT-PCR using primers for HER1: no band Normal pancreatic cells and RT-PCR using primers for HER2: band Normal pancreatic cells and RT-PCR using primers for HER3: no band Normal pancreatic cells and RT-PCR using primers for HER4: no band Pancreatic cells not expressing protein X and RT-PCR using primers for HER1: no band Pancreatic cells not expressing protein X and RT-PCR using primers for HER2: band Pancreatic cells not expressing protein X and RT-PCR using primers for HER3: band Pancreatic cells not expressing protein X and RT-PCR using primers for HER4: no band Pancreatic cells expressing protein X and RT-PCR using primers for HER1: no band Pancreatic cells expressing protein X and RT-PCR using primers for HER2: band Pancreatic cells expressing protein X and RT-PCR using primers for HER3: band Pancreatic cells expressing protein X and RT-PCR using primers for HER4: no band

Figure 1.2

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3. Western blot

"We isolated proteins from the cells, ran protein samples out on 8 percent polyacrylamide gels, and transferred the proteins to nitrocellulose membranes for blotting with HER-specific antibodies. Based on the RT-PCR data, which antibody or antibodies do you think we tested?"

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Question 1.2

a. α-HER1 antibody	Fd/yhwSeUQ6ZqQj4
b. α-HER2 antibody	uS+xJY+rPntALoOB
c. α-HER3 antibody	uS+xJY+rPntALoOB
d. α-HER4 antibody	Fd/yhwSeUQ6ZqQj4

Table

Correct.

"Since the RT-PCR data showed expression of HER2 and HER3, we performed two Western blots, one using the α-HER2 antibody and the other using the α-HER3 antibody. The expected molecular weight of HER2 is approximately 138 kDa, and the expected molecular weight of HER3 is about 135 kDa."

Western blot using the anti-HER2 antibody shows the following lanes and bands: Normal pancreatic cells: band approximately 138 kDa Pancreatic cells not expressing protein X: band approximately 138 kDa Pancreatic cells expressing protein X: band approximately 138 kDa Western blot using the anti-HER3 antibody shows the following lanes and bands: Normal pancreatic cells: band approximately 135 kDa Pancreatic cells not expressing protein X: band approximately 135 kDa Pancreatic cells expressing protein X: band approximately 135 kDa

Figure 1.3

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Figure 1.4

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Question 1.3

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Question 1.4

Which two experiments together would reveal whether HER2 and HER3 are forming heterodimers?

a. First experiment:	npEGg1AcXlUyfFamvOgfqOH5g9UpDptS3sNj8lUXmRtcGJuN0pXcrjAQpbhiHbQywV9Wy8MDRKQFXZsukvvFiR6iaO7N72Mmk1NTOfhiOY370ZvDZQntNQ==
b. Second experiment:	+5wnXOqOh5rJtfQ/XJ7z5UgcZNMcONCLZGtJC9vXvi5PlvoUSXJI7WjGXMqdIXX+qLDXEfgFJF2osJsB5U139Wvew8c5BFzNElAQUicrt7nlJx4KhERGVg==

Table

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4. Immunoprecipitation

Question 1.5

VW2OIe0anacehGrq7fcjjzdmg7O+2VjelgcpoELdCvIhhuLycLcqGzfIkhnlwNg07vB3TtEG2iFi2pc45/xh9WFgycka1UrBX/WsuPhMOYYbr7cWEHJ/4m0nSLoNQP2/gTwT8nx1/Gi2i1/7O7Ga8Kx65IkkU/lypLO53v+tqYP+qwF9mN+J/+lMZgCxxVdMTHCyXg80fQ2uVH1FtHX2f3cVmgZAblRVuM47G+ncuyFwaON/eNKLeIWKXddze/BmU9v50IDzym4fcnO11o5bKeF+LeApXQso+ccad3LBrAD7CHldNv5S6VoMV6CnIWzNzLQg2V3eNMR866zI4bJ0eUQqpa99L/pU6n6avbnouU5kZ5myuW96VGELpgnoYAfU/lK9UEPpD9jS7ohd1QTU3ABPYeD5qGvcUmPkSX/pM0CVOUsq1rZg5GoaaLi8kEIP825GFWHz0uZjtB77VH4q0a5Vrnndw2P6vsLYwsdQRvxB0b4JLZOGuzd5k9UQk68lqK+I6A5sPqXq0XMJZru49fXo7gX0Dqag

Correct.

"We isolated proteins from the cells and used either the α-HER2 antibody or the α-HER3 antibody for immunoprecipitation. Each precipitated protein fraction was run on 8 percent polyacrylamide gels, and proteins were transferred to nitrocellulose membranes for blotting with either the α-HER2 antibody or the α-HER3 antibody. Here are the data."

Immunoprecipitation using the anti-HER2 antibody, followed by Western blot using the anti-HER2 antibody shows the following lanes and bands: Normal pancreatic cells: band approximately 138 kDa Pancreatic cells not expressing protein X: band approximately 138 kDa Pancreatic cells expressing protein X: band approximately 138 kDa Immunoprecipitation using the anti-HER2 antibody, followed by Western blot using the anti-HER3 antibody shows the following lanes and bands: Normal pancreatic cells: no band Pancreatic cells not expressing protein X: no band Pancreatic cells expressing protein X: no band Immunoprecipitation using the anti-HER3 antibody, followed by Western blot using the anti-HER3 antibody shows the following lanes and bands: Normal pancreatic cells: band approximately 135 kDa Pancreatic cells not expressing protein X: band approximately 135 kDa Pancreatic cells expressing protein X: band approximately 135 kDa Immunoprecipitation using the anti-HER3 antibody, followed by Western blot using the anti-HER2 antibody shows the following lanes and bands: Normal pancreatic cells: no band Pancreatic cells not expressing protein X: no band Pancreatic cells expressing protein X: no band

Figure 1.5

Incorrect.

Figure 1.6

Try Again.

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5. Immunofluorescence microscopy

"We used fluorescein-conjugated antibodies for HER2 and a rhodamine-conjugated antibody for HER3. Here are the data."

Immunofluorescence using fluorescein-conjugated antibodies for HER2 shows the following: Normal pancreatic cells: green fluorescence around the perimeter of the cell Pancreatic cells not expressing protein X: green fluorescence around the perimeter of the cell Pancreatic cells expressing protein X: green fluorescence around the perimeter of the cell Immunofluorescence using rhodamine-conjugated antibodies for HER3 shows the following: Normal pancreatic cells: no fluorescence Pancreatic cells not expressing protein X: red fluorescence around the perimeter of the cell Pancreatic cells expressing protein X: red fluorescence around the perimeter of the cell

Figure 1.7

Question 1.6

88CMX5ZWbqN91I4jLmqs+ptkUB0RsMztvOhEXLBBOxHzxtTMLP2PnO50jfKTFlPaBGl2ARKpSKapTKZz2cDoWb6S9T6EAx/VQf0DyxHe3nsW6xpUQ+8pKmgvCnhoYAR9s8i2KhdRMaSCITiIcKKlniFITvE3rriG/gOv/BuWPUEeas3y

The RT-PCR data show expression of HER2 mRNA in all cell types and of HER3 mRNA in both cancer cell types.

Western blotting shows the same pattern of expression: HER2 protein in all cell types, HER3 protein only in cancer cell types.

Co-immunoprecipitation does not reveal any interactions between HER2 and HER3 monomers.

Immunofluorescence microscopy shows that each receptor is present on the plasma membranes of the cell types in which it is expressed.

Question 1.7

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

Correct.

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1.3 Tom's lab: G protein-coupled receptor pathways

Tom's lab: G protein-coupled receptor pathways

Kaitlin next stops by Tom's lab. Tom reports:

"We've looked at some of the G protein–coupled receptors in all of the cell types. Adrenergic receptors are expressed in the cells, and it seems that they preferentially have the β-adrenergic receptor and also have cytoplasmic cyclic AMP. Let's think about what may be going on."

true

1. PCR

"We did not do PCR, but we did do RT-PCR assays."

true

2. RT-PCR

"We isolated mRNA from the cells, made cDNA using a reverse transcriptase protocol, and then used GPCR-specific primers in an RT-PCR assay. Here are the data."

RT-PCR results show the following lanes and bands: Normal pancreatic cells and RT-PCR using primers for α1-adrenergic receptor: no band Normal pancreatic cells and RT-PCR using primers for α2-adrenergic receptor: no band Normal pancreatic cells and RT-PCR using primers for β-adrenergic receptor: no band Normal pancreatic cells and RT-PCR using primers for acetylcholine receptor: no band Pancreatic cells not expressing protein X and RT-PCR using primers for α1-adrenergic receptor: no band Pancreatic cells not expressing protein X and RT-PCR using primers for α2-adrenergic receptor: no band Pancreatic cells not expressing protein X and RT-PCR using primers for β-adrenergic receptor: band Pancreatic cells not expressing protein X and RT-PCR using primers for acetylcholine receptor: no band Pancreatic cells expressing protein X and RT-PCR using primers for α1-adrenergic receptor: no band Pancreatic cells expressing protein X and RT-PCR using primers for α2-adrenergic receptor: no band Pancreatic cells expressing protein X and RT-PCR using primers for β-adrenergic receptor: band Pancreatic cells expressing protein X and RT-PCR using primers for acetylcholine receptor: no band

Figure 1.8

true

3. Western blot

"We isolated proteins from the cells, ran protein samples out on 8 percent polyacrylamide gels, and transferred the proteins to nitrocellulose membranes for blotting with GPCR-specific antibodies. Based on the RT-PCR data, which antibody or antibodies do you think we tested?"

true

Question 1.8

a. α-α1-ad antibody	Fd/yhwSeUQ6ZqQj4
b. α-α2-ad antibody	Fd/yhwSeUQ6ZqQj4
c. α-β-ad antibody	uS+xJY+rPntALoOB
d. α-ACR antibody	Fd/yhwSeUQ6ZqQj4

Table

Correct.

"Since the RT-PCR data showed expression of the β-adrenergic receptor, we performed a Western blot using the α-β-ad antibody. The expected molecular weight of the β-adrenergic receptor is approximately 48 kDa."

Western blot using the anti-β-adrenergic receptor antibody shows the following lanes and bands: Normal pancreatic cells: no band Pancreatic cells not expressing protein X: band approximately 48 kDa Pancreatic cells expressing protein X: band approximately 48 kDa

Figure 1.9

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Figure 1.10

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Question 1.9

AQ+LURDjSHdn01GNMR+/dnVXhqp6CaS0UwcAPQc7drcCcMV9kdCT3tUc40TR1iDFrozWBOQNzHZoF7SU9kZOt+g9AoSYZ85NV1SP5bWRj1D75cx7ZhqAcIEQTmG0i1RzjL7vFHXnJqMiDITkA/TWjs665/Cha2z1VZNACwZBUoGv4SnNn1+q5f9qpkZ/SelLl6bCwc849ZzmOfNF6SzBcyYuzd3whOFsuz6MmJfwklJ0j+F5fHtLM0EFCOIIpophtpJKVseKcJ8f6DVuTWQKUSbkBlVUqFwQuLENn5W3iGqO/173bLYap9ZzNegnVyPo2y6NY4fc9titS3f53LFFDcT+n9CjMq3DRrrL2Dhg/IaxnXsEKoOmjW23p5MUnw63+Hbu+I+AZPHohhU7041Qe0PdDzq5McsBNtMOeWVq6zvQzWJoDcLXsXLOFbUKlmuVkaKS8/QzQWuqsQQlfTIYzsWF4z923iYu5o3ksWaozb9lCrZU

Correct.

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true

4. Immunoprecipitation

We did not do any immunoprecipitation experiments, but we did an experiment in which we cultured the cells in the presence of the ligand epinephrine.”

Question 1.10

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true

5. Immunofluorescence microscopy

"In the epinephrine cultures, we used a fluorescein-conjugated antibody for immunolocalization of CREB. Here are the data."

Immunofluorescence using fluorescein-conjugated antibodies for CREB shows the following: Normal pancreatic cells in the absence of epinephrine: green fluorescence in the cytosol of the cell Pancreatic cells not expressing protein X in the absence of epinephrine: green fluorescence in the cytosol of the cell Pancreatic cells expressing protein X in the absence of epinephrine: green fluorescence in the cytosol of the cell Normal pancreatic cells in the presence of epinephrine: green fluorescence in the cytosol of the cell Pancreatic cells not expressing protein X in the presence of epinephrine: green fluorescence in the nucleus of the cell Pancreatic cells expressing protein X in the presence of epinephrine: green fluorescence in the nucleus of the cell

Figure 1.11

Question 1.11

dSBMIG+5zaB5AoEjhtosTVCll/Czd25/J2urBWLVBjAYbpKcT6JFgd5HX7esFw6mxp3CyG9thM6mJK4jqlMoiaojh22af9QPCiDtNifJeY8A+vf5JQHJPWm90yNKcIEjdalebA1dtrkBEEozImdu0cYvq9BbKrllarNn5Y8OPuVdw/M4

Correct.

Activated transcription factors would be expected to be found in the nucleus.

Incorrect.

Activated transcription factors would be expected to be found in the nucleus.

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1.4 Dan's lab: TGF-β signaling pathways

Dan's lab: TGF-β signaling pathways

Kaitlin next stops by Dan's lab. Dan reports:

"Both types of pancreatic cancer cells look like they're expressing TGF-β receptor mRNA, as determined by RT-PCR, and TGF-β receptor protein, as determined by Western blotting. Immunolocalization shows the receptor present on the cell surface. Cells from normal pancreatic tissue don't express TGF-β receptor mRNA or protein."

Question 1.12

9SSBnGe7URAHnfo3bKx5yjUVxv8iTrqv1vDBIsyLew/JIYKxyJxtICRhTt/rcmCfLc/+mwRBFpNU/75KBgEhgud3n5ohGX/Q8bdo32j220ZlbeWqW/loO0XprUq8H/92eQ6HApQGkWy2CtUtNiLVOXTyRIFqOQSRSjzNL1Bj9/Va+tfTEJGy6NDHqIBHEJKTPaduIuaMtsU6LpV8fWYdEVb7vk2siDFBaa6sEkmilC0ehjy2IrfxwvXwaZsb6VX3gu1fJWFnAZZYKmYbUaxMj22ovai+WaMTMnWAU1rKoLY6nbcT34J/aRbnvjMHV5T5D8799iOlNXLpB4XBLEycmEqRX+JvXQm7JYbQHeTF9YqKJDhi6mtNlMCcnGLh11ECec5+AutkE42lToCSasU8H5YEp/Q3JbHBZ/FYTCYXljKVIxANfqWGorvEiWPQlpUIlxguztX4xGF48drlargL1c5wzgoqVYl/x3AIaE7rZfN8MfStRzeFk8l3eWJI7DnAkRm0DzU81zv3ge24sQIAHzkXzCLuWrxGYPM+MZ1k0Ccp8AOM3PiALfX3kglxSdwqMDEBdxpk1x5zA4rHXB3tBfyCqDmzsu2HEojmS4A9MBps8Q+HorXIWE9u8Zp4Gpz2gJYjr10BOA1qjyOGmBAblUwJwFltqp3khE2gX0LCpi80VNM5p6u02PVFiwIpcAvX9sSPiJFwdmCDrCap2Wxgp2z8PD5cEMt65A0uoomm86Uke5zecWdG6PyknhBU2fDd33YVGHQoJjo=

Correct.

Incorrect.

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Question 1.13

Dan shows Kaitlin the data from cells that were cultured in the presence or absence of TGF-β, then exposed to a fluorescein-conjugated antibody for immunolocalization of Smad:

Immunofluorescence using fluorescein-conjugated antibodies for Smad shows the following: Normal pancreatic cells in the absence of ligand: green fluorescence in the cytosol of the cell Pancreatic cells not expressing protein X in the absence of ligand: green fluorescence in the cytosol of the cell Pancreatic cells expressing protein X in the absence of ligand: green fluorescence in the cytosol of the cell Normal pancreatic cells in the presence of TGF-β: green fluorescence in the cytosol of the cell Pancreatic cells not expressing protein X in the presence of TGF-β: green fluorescence in the nucleus of the cell Pancreatic cells expressing protein X in the presence of TGF-β: green fluorescence in the nucleus of the cell

Figure 1.12

Pg2/ZZGqZGdEBaufE3h2ooCzZdGtGNj/lsLuP/mciwXhN7Ko

Adding the ligand TGF-β to cells that express the TGF-β receptor causes the redistribution of the Smad transcription factor to the nucleus, as expected.

1.5 Sarah's lab: Wnt signaling pathways

Sarah's lab: Wnt signaling pathways

Kaitlin next stops by Sarah's lab. Sarah reports:

"We've done characterizations of Frizzled-2 and its co-receptor LRP5 in the different cell lines. Here are the data."

Western blot using the anti-Fz2 antibody shows the following lanes and bands: Normal pancreatic cells: no band Pancreatic cells not expressing protein X: no band Pancreatic cells expressing protein X: band approximately 63 kDa Western blot using the anti-LRP5 antibody shows the following lanes and bands: Normal pancreatic cells: band approximately 179 kDa Pancreatic cells not expressing protein X: band approximately 179 kDa Pancreatic cells expressing protein X: band approximately 179 kDa

Figure 1.13

Question 1.14

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

Correct.

Incorrect.

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Question 1.15

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Treat cells with Wnt and use immunofluorescence microscopy to see if β-catenin is localized to the nucleus. In addition, the phosphorylation status of β-catenin could be determined with a phospho-specific antibody and Western blotting.

Question 1.16

Performing the immunolocalization experiment gives the results below.

Immunofluorescence using fluorescein-conjugated antibodies for β-catenin shows the following: Normal pancreatic cells in the absence of ligand: green fluorescence in the cytosol of the cell Pancreatic cells not expressing protein X in the absence of ligand: green fluorescence in the cytosol of the cell Pancreatic cells expressing protein X in the absence of ligand: green fluorescence in the cytosol of the cell Normal pancreatic cells in the presence of Wnt ligand: green fluorescence in the cytosol of the cell Pancreatic cells not expressing protein X in the presence of Wnt ligand: green fluorescence in the cytosol of the cell Pancreatic cells expressing protein X in the presence of Wnt ligand: green fluorescence in the nucleus of the cell

Figure 1.14

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Correct.

Incorrect.

Question 1.17

3GbI96NaQ3n1Kl52JfEyYS4Ir1+V3wp1HrOIghvAOA2CLKhA5WDtR9XiGN7b8HBMUaUfbqNyT+4QGmYrEhWmtWUkyoi6R2ZTScwbBnHCBV/YtaapuqpGYXbfIhPI10xjVNj3crw0TIUVMJ2GvrNfICJ39A0lKZI6WMz417rG+BRZo8z9iLL036T2uPffs1jaNhAecY35IoEodcwvrx3GHnqj27zLn9ur965/nBE7KqiM6OzXLEz7KA==

You could treat cells with the following ligands to test for the induction of protein expression:

NRG1 for HER2-HER3 signaling
Epinephrine for β-adrenergic receptor–mediated signaling
TGF-β for TGF-β receptor–mediated signaling
Wnt ligand for Fz2-LRP5 signaling

The treated cells could be cultured, and the secreted proteins in the culture medium could be assayed by polyacrylamide gel electrophoresis and Coomassie blue staining to identify a 90-kDa protein product, protein X.

Question 1.18

Coomassie staining of protein extracts from cells treated with the following ligands showed: Normal pancreatic cells in the presence of NRG1: standard smear pattern Normal pancreatic cells in the presence of epinephrine: standard smear pattern Normal pancreatic cells in the presence of TGF-β: standard smear pattern Normal pancreatic cells in the presence of Wnt: standard smear pattern Pancreatic cells not expressing protein X in the presence of NRG1: standard smear pattern Pancreatic cells not expressing protein X in the presence of epinephrine: standard smear pattern Pancreatic cells not expressing protein X in the presence of TGF-β: standard smear pattern Pancreatic cells not expressing protein X in the presence of Wnt: standard smear pattern Pancreatic cells expressing protein X in the presence of NRG1: standard smear pattern Pancreatic cells expressing protein X in the presence of epinephrine: standard smear pattern Pancreatic cells expressing protein X in the presence of TGF-β: standard smear pattern Pancreatic cells expressing protein X in the presence of Wnt: standard smear pattern with an extra band approximately 90 kDa

Figure 1.15

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Correct.

Incorrect.

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1.6 Conclusion

Conclusion

Question 1.19

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Activation of the Wnt signaling pathway results in persistent nuclear localization of β-catenin, which would lead to constitutive expression of genes including the gene encoding protein X. Inappropriate activation of the Wnt pathway is characteristic of many human cancers. In many tumors, the level of free β-catenin is abnormally high, and this observation provided one of the earliest clues that β-catenin can activate many growth-promoting genes. Inactivating mutations in genes encoding APC and Axin are found in multiple types of human cancers, as are mutations in β-catenin phosphorylation sites for GSK3 or CK1; these mutations reduce formation of the cytosolic complex (Figure 16-30a), reduce β-catenin degradation, and allow β-catenin to activate gene expression in the absence of normal Wnt signaling (page 753).

Question 1.20

X2zlzF4FUKDCcUGJ76IYj9ATh1DQz/WBRQ1Xh6X4B6QCQyKsrv2ZTVUE8PjrrlNN+LmY4sIRyPBHkRpUU2mam5x/nTlbdszVz++QaxAp9tblZdjPr4Z4mnX6iaPL57Nlwi8kFlyuooP8WGNisaWnPULtZRj9z+9U9+xMskiZeTaPk7gCo5yxDKPHbee+EUiumSC/z+B/IXFU8TsL9RTI6+qMNidIdcE0vv850Ac+qFeGBC3jsjPnuvQG24AJtDb1cNI7hI1lOcH2MvYcMPbiOGrZ4aS+S6EJscBt52w86D+JKWvHuspPWi5t92o9EHl1eqe3aohfrF52vdGtTULMq8sWVPTi8Iej+rX7cQtpfSc6iRQ80Gqs4C0okmf+NXgG2bGBmTiETlypboiJM/psXMBEtGisqrMmzF9gZ4sxh5r9HBMI6rCVvOaJUK29wWWOtRczBA==

Correct.

β-Catenin activity is regulated by phosphorylation at serine and threonine residues through GSK3 and CK1. A reduction or loss in phosphorylation would result in an increased distribution of β-catenin in the nucleus.

Incorrect.

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Question 1.21

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Correct.

NRG2 would stimulate cell signaling through the HER2-HER3 complex. The aggressive cancer cells appear to have a primary defect in Wnt signaling, which leads to chronic activation of HER2-HER3 signaling.

Incorrect.

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Question 1.22

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

Correct.

Incorrect.

Try Again.

Question 1.23

tBUU36HqgkxqH6ojWjuYhMuPkF2Gv3jVVft8csKSbycLHPZBFdz09FawADchWH9dEnoAWgRaRmIZz1JD

These aggressive cancer cells appear to have a primary defect in Wnt signaling, which leads to constitutive β-catenin activity as a transcription factor. As a result, NRG2 is continuously secreted, leading to activation of the MAP kinase pathway through HER2-HER3. Drugs that target the β-catenin pathway would be most therapeutic, as they would reduce the contributions of Wnt and HER2-HER3 signaling to the cancer. In theory, siRNA approaches used to degrade NRG2 mRNA would also reduce the autocrine effects of secreted NRG2.

Activity results are being submitted...

1.7 Additional Information

Additional Information: Stages of FDA Approved Drug Development

FDA Drug Development Process http://www.fda.gov/ForPatients/Approvals/Drugs/default.htm

Step 1: Discovery and Development

Discovery of new drugs can arise from (1) new information about a disease process, (2) testing the therapeutic effects of compounds against many diseases, (3) unexpected effects of current treatments, and (4) new technologies for drug delivery.

Drug development includes determination of (1) mechanisms of absorption, metabolism, and excretion, (2) potential benefits, (3) optimum dosage, (4) optimum delivery, (5) side effects, (6) effects on different groups of people, (7) interactions with other drugs, and (8) comparative effectiveness.

For more information visit: http://www.fda.gov/ForPatients/Approvals/Drugs/ucm405382.htm

Step 2: Preclinical Research

In vitro (cell lines) and in vivo (animal) research is carried out to determine dosing and toxicity levels. Findings are reviewed to determine whether the drug should be tested in humans.

For more information visit: http://www.fda.gov/ForPatients/Approvals/Drugs/ucm405658.htm

Step 3: Clinical Research

The drug is tested in clinical trials in humans. Several factors are important in the design of these clinical trials: (1) selection criteria, (2) number of participants, (3) study length, (4) presence of a control group, (5) drug route and dosage, (6) type of data to be collected, and (7) data analysis plan. Trials consist of four levels, or phases, starting with a small-scale approach and moving to a large-scale approach.

For more information visit: http://www.fda.gov/ForPatients/Approvals/Drugs/ucm405622.htm

Step 4: FDA Review

Drug developers submit data on preclinical and clinical trials and can file an application to market the drug. The FDA reviews the information and decides whether to give approval or not.

For more information visit: http://www.fda.gov/ForPatients/Approvals/Drugs/ucm405570.htm

Step 5: FDA Post-Market Safety Monitoring

The FDA examines problems associated with a drug over its lifetime and can issue warnings or changes depending on the severity of the problems.

For more information visit: http://www.fda.gov/ForPatients/Approvals/Drugs/ucm405579.htm