Question

Those who inherit a mutant allele of the \(R B 1\) tumor-suppressor gene are at risk for developing a bone cancer called osteosarcoma. You suspect that in these cases, osteosarcoma requires a mutation in the second \(R B 1\) allele, and you have cultured some osteosarcoma cells and obtained a cDNA clone of a normal human \(R B 1\) gene. A colleague sends you a research paper revealing that a strain of cancer-prone mice develops malignant tumors when injected with osteosarcoma cells, and you obtain these mice. Using these three resources, indicate which experiments you would perform to determine the following: (a) whether osteosarcoma cells carry two \(R B 1\) mutations, (b) whether osteosarcoma cells produce any pRB protein, (c) if the addition of a normal \(R B 1\) gene will change the cancercausing potential of osteosarcoma cells.

Short answer

Answer: The three experiments performed are: (a) Experiment A aims to determine if osteosarcoma cells carry two \(R B 1\) mutations by sequencing the \(R B 1\) genes in these cells. (b) Experiment B aims to determine if osteosarcoma cells produce any pRB protein using a Western blot. (c) Experiment C aims to determine if the addition of a normal \(R B 1\) gene can change the cancer-causing potential of osteosarcoma cells by comparing tumor growth and survival rates in cancer-prone mice injected with transfected and control osteosarcoma cells.

Step-by-step solution

Experiment A: Determine if osteosarcoma cells carry two \(R B 1\) mutations

To determine if osteosarcoma cells carry two \(R B 1\) mutations, we need to sequence the \(R B 1\) genes in these cells. First, extract the genomic DNA from the osteosarcoma cells. Then, amplify the \(R B 1\) gene region in the extracted genomic DNA using polymerase chain reaction (PCR). After that, sequence the PCR products to reveal any mutations in the \(R B 1\) gene.

Experiment B: Determine if osteosarcoma cells produce any pRB protein

To determine if osteosarcoma cells produce any pRB protein, we first need to prepare total protein extracts from the cells. Afterward, perform a Western blot using an antibody specific for pRB and appropriate secondary antibodies to detect its presence. A positive Western blot result would indicate the presence of pRB protein, whereas a negative result would indicate that osteosarcoma cells do not produce pRB protein.

Experiment C: Determine if a normal \(R B 1\) gene can change the cancer-causing potential of osteosarcoma cells

First, transfect the osteosarcoma cells with the normal human \(R B 1\) cDNA clone to create osteosarcoma cells that overexpress the normal \(R B 1\) gene. Verify the overexpression of \(R B 1\) through quantitative reverse transcription PCR (qRT-PCR) or Western blot. Next, inject both the transfected osteosarcoma cells and control (untransfected) osteosarcoma cells into separate groups of cancer-prone mice. Monitor tumor growth and survival rates in both groups of mice. A decrease in tumor growth or an increase in survival rate in mice injected with transfected osteosarcoma cells, compared to the control group, would suggest that adding a normal \(R B 1\) gene can change the cancer-causing potential of osteosarcoma cells.

Key concepts

Osteosarcoma

Osteosarcoma is a type of bone cancer that is most prevalent among teenagers and young adults. It typically arises in the osteoblast cells, which are responsible for forming new bone. This cancer often affects the long bones in the arms and legs, particularly around the knees.
The cause of osteosarcoma is linked to genetic factors such as mutations in certain genes. Symptoms include pain in a bone or joint, swelling, and sometimes a noticeable lump. Early detection and treatment are crucial to managing osteosarcoma effectively.

• Common sites: long bones, but can occur in any bone.
• Symptoms: pain, swelling, lump formation.
• Common in: adolescents and young adults.

RB1 Gene

The RB1 gene provides instructions for making a protein known as pRB, which plays a critical role as a tumor suppressor. Tumor suppressor genes are like brakes on cell division, ensuring cells do not divide uncontrollably.
The RB1 gene, when functioning correctly, helps prevent the formation of tumors by controlling the cell cycle. However, mutations in this gene can lead to a loss of this control, which may result in cancer. In families with hereditary cancer syndromes, mutations in the RB1 gene can be passed down, increasing the risk of developing cancers like osteosarcoma.

• Function: controls cell cycle, prevents tumor formation.
• Mutations: lead to uncontrolled cell division.
• Related conditions: retinoblastoma, osteosarcoma.

Mutation Analysis

Mutation analysis involves examining specific segments of DNA to identify alterations that may be responsible for disease development. In the context of the RB1 gene, the process often starts with extracting genomic DNA from cells suspected of having mutations.
Following extraction, polymerase chain reaction (PCR) is used to amplify the gene region of interest to facilitate sequencing. Sequencing helps pinpoint exact mutations, which can inform diagnosis, prognostic evaluations, and potential treatment approaches.

• Steps: DNA extraction, PCR amplification, sequencing.
• Purpose: detect gene mutations linked to disease.
• Outcome: guides diagnosis and treatment strategies.

Western Blot

Western blot is a laboratory technique used to detect specific proteins in a tissue or cell sample. It is particularly useful for verifying the presence or absence of a protein such as pRB, which the RB1 gene codes for.
The process begins with protein extraction from the cells. The extracted proteins are then separated by size using gel electrophoresis. After transferring the proteins to a membrane, they are probed with antibodies that specifically recognize the target protein.

• Purpose: detect and quantify specific proteins.
• Steps: protein extraction, gel electrophoresis, antibody probing.
• Applications: validating expression of proteins like pRB.

Gene Transfection

Gene transfection is a method used to introduce foreign DNA into cells. This technology allows scientists to manipulate gene expression within cells, which can aid in research on genetic functions and potential therapies.
For the RB1 gene, transfection involves introducing a normal version of the gene into osteosarcoma cells. The goal is to determine if this can counteract the cell's cancerous characteristics. Successful transfection can lead to the expression of the normal gene, enabling researchers to assess its effects on tumor growth or suppression directly.

• Process: introducing new genetic material into cells.
• Applications: research, gene therapy, functional studies.
• Outcome: measure changes in cell behavior or characteristics.