Passage
Proteins known as cancer/testis antigens (CTAs) are normally expressed exclusively in germline cells but may become reactivated in cancer cells due to chromatin remodeling (eg, gene activation due to DNA demethylation) . Because of their normally restricted expression, CTAs can elicit an immune response when expressed by cancer cells derived from other tissue types. Several CTAs are known to inhibit apoptosis or promote cell division in cancer cells, making them more resistant to traditional chemotherapy. Accordingly, cancer research instead has focused on immunotherapy treatments targeting CTAs.In a study, researchers isolated peripheral blood lymphocytes (B lymphocytes, T lymphocytes, natural killer [NK] cells) from two healthy individuals. These lymphocytes were cultured with signaling factors known to promote helper T cell (T_h cell) proliferation. After 7 days, cultures from both individuals were enriched for T_h cells, which expressed proteins generally involved in activating other lymphocytes. A subset of the cultures from each individual was treated with 5-Aza-CdR, a DNA-demethylating agent. Reverse transcription polymerase chain reaction (RT-PCR) was used to analyze expression of several CTAs (ie, MAGEs) in both untreated and 5-Aza-CdR-treated lymphocyte cultures, as well as in untreated melanoma (skin cancer) and breast cancer cell lines (Figure 1) . GAPDH, a protein expressed in most cell types, served as a control.
Figure 1 Gels showing RT-PCR results for MAGE expressionAdditional peripheral blood lymphocytes were isolated from the same healthy individuals and incubated with the T_h cells that had been treated with 5-Aza-CdR. After 11 days, these cultures consisted primarily of an increased number of NK cells and cytotoxic T lymphocytes (CTLs) . Researchers found that these CTLs were able to induce apoptosis in breast cancer cell lines but did not affect healthy cells.Researchers used a similar procedure to induce CTL proliferation in lymphocytes isolated from a breast cancer patient. The CTLs were then incubated with cells obtained from a biopsy of the patient's cancerous tissue. Following this experiment, researchers found that apoptosis was induced in the patient's cancer cells.
Kirkin et al. Adoptive cancer immunotherapy using DNA-demethylated T helper cells as antigen-presenting cells. Nature Communications. 2018; 9:785
-Which of the following conclusions is best supported by the results presented in Figure 1?

Question

Passage
Proteins known as cancer/testis antigens (CTAs) are normally expressed exclusively in germline cells but may become reactivated in cancer cells due to chromatin remodeling (eg, gene activation due to DNA demethylation) . Because of their normally restricted expression, CTAs can elicit an immune response when expressed by cancer cells derived from other tissue types. Several CTAs are known to inhibit apoptosis or promote cell division in cancer cells, making them more resistant to traditional chemotherapy. Accordingly, cancer research instead has focused on immunotherapy treatments targeting CTAs.In a study, researchers isolated peripheral blood lymphocytes (B lymphocytes, T lymphocytes, natural killer [NK] cells) from two healthy individuals. These lymphocytes were cultured with signaling factors known to promote helper T cell (T_h cell) proliferation. After 7 days, cultures from both individuals were enriched for T_h cells, which expressed proteins generally involved in activating other lymphocytes. A subset of the cultures from each individual was treated with 5-Aza-CdR, a DNA-demethylating agent. Reverse transcription polymerase chain reaction (RT-PCR) was used to analyze expression of several CTAs (ie, MAGEs) in both untreated and 5-Aza-CdR-treated lymphocyte cultures, as well as in untreated melanoma (skin cancer) and breast cancer cell lines (Figure 1) . GAPDH, a protein expressed in most cell types, served as a control.

Figure 1 Gels showing RT-PCR results for MAGE expressionAdditional peripheral blood lymphocytes were isolated from the same healthy individuals and incubated with the T_h cells that had been treated with 5-Aza-CdR. After 11 days, these cultures consisted primarily of an increased number of NK cells and cytotoxic T lymphocytes (CTLs) . Researchers found that these CTLs were able to induce apoptosis in breast cancer cell lines but did not affect healthy cells.Researchers used a similar procedure to induce CTL proliferation in lymphocytes isolated from a breast cancer patient. The CTLs were then incubated with cells obtained from a biopsy of the patient's cancerous tissue. Following this experiment, researchers found that apoptosis was induced in the patient's cancer cells.
Kirkin et al. Adoptive cancer immunotherapy using DNA-demethylated T helper cells as antigen-presenting cells. Nature Communications. 2018; 9:785
-Which of the following conclusions is best supported by the results presented in Figure 1?

Quizplus · Accepted Answer

11ecba2d_1040_f622_a3bf_cb3da15df78d_MD0008_00 Reverse transcription polymerase chain reaction (RT-PCR) is used to analyze cells for the presence of specific mRNA transcripts (ie, expressed genes). The enzyme reverse transcriptase is used to convert mRNA to double-stranded cDNA, which is then amplified via PCR using primers specific to the transcript(s) of interest. Following RT-PCR, gel electrophoresis can be used to analyze samples for the presence or absence of specific transcripts (ie, band presence). In this experiment, researchers performed RT-PCR to test for expression of specific CTAs (MAGEs) in several cell types. Figure 1 shows the results of this RT-PCR analysis. MAGE-A10 gene expression was not detected (no band was present) in breast cancer cells, which indicates that MAGE-A10 protein is not expressed in these cells. Therefore, cytotoxic T lymphocytes (CTLs) and other immune cells would not target MAGE-A10 when interacting with these breast cancer cells. In contrast, MAGE-A10 gene expression was detected in melanoma cells. Therefore, MAGE-A10 is a potential target for immune cells interacting with these melanoma cells. Accordingly, these results best support the conclusion that MAGE protein classes targeted by immune cells will likely differ depending on the cancer cell type. (Choice A) Figure 1 shows that cDNA for different MAGE genes traveled different distances through the gels, indicating that MAGE transcripts vary in length and are not composed of the same number of nucleotides. (Choice C) Figure 1 shows that MAGE-A6 is the largest MAGE transcript (~800 bp). However, RT-PCR of a transcript with the highest expression would yield the most cDNA such that gel electrophoresis of this transcript would produce the most intense band. The intensity of the MAGE-A6 band is similar to the intensities of other MAGE bands in cancer cells, indicating that MAGE-A6 does not exhibit the highest expression. (Choice D) RT-PCR measures gene expression, not protein activity. Expression of a gene does not necessarily indicate that the protein product is active. However, the results show that more MAGE gene types are expressed in melanoma cells than in breast cancer cells. Educational objective: RT-PCR is used to analyze gene expression (ie, the presence of specific mRNA transcripts). Reverse transcriptase is used to convert mRNA into double-stranded cDNA, and PCR is performed to amplify transcripts of interest.

Passage Proteins Known as Cancer/testis Antigens (CTAs) Are Normally Expressed Exclusively