SB-BB

Passage 8

The retinoblastoma protein (pRB) has a primary role as a transcriptional coregulator of genes involved in several important cellular processes such as the cell cycle. Researchers hypothesized that pRB may also play a role in mitochondrial apoptosis. The mitochondrial apoptosis pathway involves the activation of the BAX and BAK proteins, which results in the release of the caspase activator cytochrome c. To investigate a potential role for pRB in this process, researchers used stable cell lines of wild-type (WT), BAK-deficient (Bak–/–), and BAX-deficient (Bax–/–) rat embryonic fibroblasts in which pRB expression could be induced (pRB induced) above basal levels (pRB basal). Cells with basal or induced pRB expression for 24 h were treated with the pro-apoptotic factor TNFα for 48 h. Then, the percentage of apoptotic cells in each group was determined by staining cytoplasmic phosphatidylserine with the fluorescent marker AnnexinV (Figure 1).

Figure 1 Percentage of apoptosis in each cell line after treatment with TNFα

Constructs were generated to contain inducible versions of three domains of pRB including the N-terminus (RB_N; residues 1–372), the small pocket domain (RB_SP; residues 373–766), and the C-terminus (RB_C; residues 767–928). Cells with basal levels of pRB, inducible pRB, or the three variants, RB_N, RB_SP, and RB_C, were treated with TNFα for 24 h. Then, the percentage of apoptosis was determined by AnnexinV staining (Figure 2).

Figure 2 Percentage of apoptosis in cells with different forms of RB after treatment with TNFα

Adapted from K.I. Hilgendorf et al., “The Retinoblastoma Protein Induces Apoptosis Directly at the Mitochondria.” Genes & Development ©2013 Cold Spring Harbor Laboratory Press.

Questions

57. In order to determine the effect of pRB on tumor growth, tumor cells containing no pRB (Rb–/–), basal levels of pRB (basal pRB), or additional induced amounts of pRB (induced pRB), were injected into mice. Tumor cell growth was measured at eleven days post graft. Which graphic shows the expected result of this experiment?

58. Which statement is best supported by the data shown in Figure 1?

  1. TNFα stimulates nuclear to mitochondrial translocation of pRB.
  2. pRB exerts its effects on TNFα-stimulated apoptosis via the extrinsic apoptotic pathway.
  3. TNFα-stimulated apoptosis correlates negatively with induced pRB levels in Bak–/– cells.
  4. pRB induction requires BAX, but not BAK, for TNFα-stimulated apoptosis.

59. Which experimental approach would be LEAST effective to determine the localization of pRB within a cell?

  1. Fusion of a fluorescent tag to pRB
  2. Use of a fluorescent probe that hybridizes to rb transcripts
  3. Cellular fractionation to isolate different organelles
  4. Pull down of pRB and identification of interacting proteins by mass spectrometry

60. Rhodamine 123 is a fluorescent dye that binds to polarized membranes and is used to label mitochondria. A cell line expressing basal levels of pRB or which form of induced pRB would most likely exhibit the lowest level of rhodamine 123 staining?

  1. pRB induced
  2. RB_N
  3. RB_SP
  4. RB_C

61. The caspase activator released during the mitochondrial apoptotic pathway primarily functions in which cellular process?

  1. Proton pumping
  2. Heme biosynthesis
  3. Calcium signaling
  4. Electron transport

62. Cells were incubated with the indicated amounts of recombinant pRB only, or recombinant pRB in the presence of BAX and BAK. Following centrifugation, cell membranes were disrupted but organelles remained intact. Levels of cytochrome c in the supernatant (SN) and pellet fractions were analyzed by Western blot. Which graphic shows the expected result?

Answers

Question 57 Solution: The correct answer is C. This is a Biology question that falls under content category “Transmission of genetic information from the gene to protein.” The answer to this question is C because the data in the passage show that pRB has a pro-apoptotic function, which appears to work in a dose-dependent manner. Based on this, you would expect that induction of pRB would decrease tumor cell growth by promoting apoptosis. This would decrease overall tumor growth and volume. Tumor cells with basal levels of pRB are expected to have less growth than tumor cells that do not contain pRB. This is a Scientific Reasoning and Problem Solving question as you are predicting the outcome of an experiment.
Question 58 Solution: The correct answer is D. This is a Biochemistry question that falls under content category “Processes of cell division, differentiation, and specialization.” The answer to this question is D because pRB induction enhanced TNFα-stimulated apoptosis in both WT and Bak–/– cells. However, there was no effect in Bax–/– cells, indicating that BAX is required for TNFα-stimulated apoptosis. This is a Data-based and Statistical Reasoning question as it requires interpretation of the data shown in Figure 1.
Question 59 Solution: The correct answer is B. This is a Biology question that falls under content category “Assemblies of molecules, cells, and groups of cells within single cellular and multicellular organisms.” The answer to this question is B because tagging the rb transcript would be the least helpful since this is expected to be found in the cytoplasm where the ribosomes are located. This is a Reasoning about the Design and Execution of Research question as it requires you to understand the different compartments present in a eukaryotic cell and reason about the experimental approaches that could help determine where pRB is localized.
Question 60 Solution: The correct answer is C. This is a Biochemistry question that falls under content category “Principles of bioenergetics and fuel molecule metabolism.” The answer to this question is C because when apoptosis is induced by pRB, rhodamine 123 would no longer be able to label the mitochondria efficiently due to the loss of membrane potential. Figure 2 shows that the number of apoptotic cells is highest when an inducible copy of RB_SP is expressed, indicating that this would likely result in the lowest amount of mitochondrial labeling. This is a Data-based and Statistical Reasoning question because it requires the interpretation of the data presented in Figure 2.
Question 61 Solution: The correct answer is D. This is a Biochemistry question that falls under content category “Principles of bioenergetics and fuel molecule metabolism.” The answer to this question is D because cytochrom c is associated with the mitochondrial inner membrane, where it functions in the electron transport chain. This is a Knowledge of Scientific Concepts and Principles question because it requires assumed knowledge of biochemical metabolic processes.
Question 62 Solution: The correct answer is A. This is a Biochemistry question that falls under content category “Processes of cell division, differentiation, and specialization.” The answer to this question is A because cytochrome c is present in the mitochondrial intermembrane space during normal cell function and is released into the cytoplasm when apoptosis is initiated. From the passage it is shown that pRB is able to induce apoptosis in a BAX dependent manner. This indicates that you would only expect to see cytoplasmic cytochrome c present in the supernatant when both BAX and RB are present as illustrated in graphic A. This is a Scientific Reasoning and Problem Solving question as you are predicting the outcome of an experiment.