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Abstract

  1. Top of page
  2. Abstract
  3. THE EXPERIMENT
  4. CORRECT ANSWERS
  5. EXPLANATIONS
  6. REFERENCE

Terms to be familiar with before you start to solve the test: Tumor suppressor proteins; p53 protein; p53-positive and p53-negative tumors; transcription factors; protein phosphorylation; retinoblastoma protein; Go phase; apoptosis; chaperone; transfection; expression plasmid; cDNA; papillomavirus; Western blot analysis; γ-irradiation; mitotic index; [3H]thymidine labeling.


THE EXPERIMENT

  1. Top of page
  2. Abstract
  3. THE EXPERIMENT
  4. CORRECT ANSWERS
  5. EXPLANATIONS
  6. REFERENCE

The p53 tumor suppressor protein is an important regulator of cell fate.

Multiple Completion

A question or incomplete statement is followed by four numbered completions, one or more of which are correct. Select

  • A if 1, 2, and 3 are correct;

  • B if 1 and 3 are correct;

  • C if 2 and 4 are correct;

  • D if only 4 is correct;

  • E if all four are correct.

1. Which of the following statements describes the features of p53?

  • It is a transcription factor.

  • Its main function is to phosphorylate the retinoblastoma protein.

  • Cells enter the Go phase upon p53 action.

  • It has anti-apoptotic effects.

Tumors often carry p53 mutations, whereas in normal cells the level of p53 protein is under tight control exerted by several mechanisms. The following test deals with the role of two proteins in this process. The enzyme NAD(P)H:quinone oxido-reductase (NQO)111 is involved in the redox regulation of the cells, whereas heat shock protein (Hsp)90 is a chaperone; both are important p53 regulators.

p53-Negative human colon carcinoma cells were transfected with expression plasmids carrying the cDNAs of p53, NQO1, or the papillomavirus E6 gene. Twenty-four hours after transfection, protein extracts were prepared from the cells and Western blot analyses were performed using the appropriate antibodies. cDNA combinations used for transfection and the results of Western blotting are shown in Fig. 1.

In the second experiment, two inhibitors were tested for their effects on γ-irradiated myeloid leukemia cells: the NQO1 inhibitor dicoumarol and the Hsp90 inhibitor radicicol. Western blots using anti-p53 and anti-actin antibodies are shown in Fig. 2.

Study the figures and solve the following multiple-choice questions.

Experiment Analysis

The following statements are related to the information presented in the description of the experiment. Based on the information given, select

  • A if the statement is supported by the information given;

  • B if the statement is contradirected by the information given;

  • C if the statement is neither supported nor contradicted by the information given.

  • 2. The expression of actin gene is under p53 regulation.

  • 3. Papillomavirus E6 oncoprotein induces apoptosis of human colon carcinoma cells transfected with p53 cDNA.

  • 4. NQO1 forms a complex with the E6 protein in colon carcinoma cells.

Four-choice Association

In this type of question, a set of lettered headings is followed by a list of numbered words or phrases. Select

  • A if the word or phrase is associated with A only;

  • B if the word or phrase is associated with B only;

  • C if the word or phrase is associated with A and B;

  • D if the word or phrase is associated with neither A nor B.

  • A: NQO1 enzyme

  • B: Hsp90 protein

  • C: Both of them

  • D: Neither of them

  • 5. Its activation stimulates p53 breakdown.

The following problems [6–9] were not studied in this experiment, but correct conclusions can be drawn from knowing the mechanism of action of the p53 protein.

  • 6. It causes the accumulation of DNA damage in the cells.

  • 7. Enhanced activity of this enzyme increases the mitotic index of the cell population.

  • 8. It stimulates the phosphorylation of retinoblastoma protein.

  • 9. Stimulates [3H]thymidine labeling of the nuclei of leukemia cells.

Relationship Analysis

This type of question consists of a sentence with two main parts: an assertion and a reason for that assertion. Select

  • A if both assertion and reason are true statements and the reason is a correct explanation of the assertion;

  • B if both assertion and reason are true statements, but the reason is not a correct explanation of the assertion;

  • C if the assertion is true, but the reason is a false statement;

  • D if the assertion is false, but the reason is a true statement;

  • E if both assertion and reason are false statements.

  • 10. Radicicol can be used as a powerful chemotherapeutic drug to treat p53-positive myeloid leukemia patients, because it increases the apoptosis-inducing effect of radiotherapy.

CORRECT ANSWERS

  1. Top of page
  2. Abstract
  3. THE EXPERIMENT
  4. CORRECT ANSWERS
  5. EXPLANATIONS
  6. REFERENCE

1. B 6. D

2. B 7. D

3. B 8. D

4. C 9. D

5. D 10. E

EXPLANATIONS

  1. Top of page
  2. Abstract
  3. THE EXPERIMENT
  4. CORRECT ANSWERS
  5. EXPLANATIONS
  6. REFERENCE

The p53 protein is a transcription factor that is able to suppress malignant transformation of cells by several mechanisms. It induces 1) proteins involved in DNA repair to eliminate DNA damage; 2) cyclin-dependent kinase (Cdk) inhibitors to evoke quiescence (Go phase); and 3) pro-apoptotic proteins to kill cells with unrepairable DNA damage (MCQ1: B). The actin gene is not among those genes regulated by p53 (compare samples 2 to 5 in Fig. 1; MCQ2: B). The E6 oncoprotein is able to down-regulate p53 (compare samples 2 and 4 in Fig. 1), and this anti-apoptotic effect contributes to the oncogenic action of papillomaviruses (MCQ3: B). NQ01 prevents the effect of E6 (compare samples 3, 4, and 5 in Fig. 1), but possible complex formation between these two proteins was not analyzed in this experiment (MCQ4: C).

γ-Irradiation causes DNA damage and thereby increases the level of p53 in p53-positive cells (compare samples 1 and 4 in Fig. 2). The effect of dicoumarol and radicicol indicates that both NQ01 and Hsp90 is involved in the mediation of this effect (compare samples 4, 5, and 6 in Fig. 2). Both proteins are, thus, involved in DNA-damage-stimulated signaling processes leading to p53 induction (MCQ5: D). DNA repair (MCQ6: D) and inhibition of the cell cycle by blocking the phosphorylation of retinoblastoma protein by cyclin-dependent kinases (MCQ7: D, MCQ8: D, MCQ9: D). These experiments suggest that the inhibition of Hsp90 is unlikely to stop malignant cell proliferation in p53-positive myeloid leukemia patients (MCQ10: E).

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Figure Fig. 1.. Western blot analysis of human colon carcinoma cells transfected with p53, NQO1, and E6 cDNAs (for details see the text).

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Figure Fig. 2.. The effect of γ-irradiation, dicoumarol, and radicicol treatment on myeloid leukemia cells (details in the text).

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  • 1

    The abbreviations used are: NQO, NAD(P)H:quinone oxido-reductase; Hsp90, heat shock protein 90 kDa; MCQ, multiple-choice question.

REFERENCE

  1. Top of page
  2. Abstract
  3. THE EXPERIMENT
  4. CORRECT ANSWERS
  5. EXPLANATIONS
  6. REFERENCE