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
-Treating DNA with the compound bisulfite converts any unmethylated cytosines into uracils. Bisulfite sequencing, which takes advantage of this mechanism, was used to compare DNA methylation of the MAGE-A1 gene in healthy cells and melanoma cells. Based on the passage, which of the following graphs most likely represents the percentage of cytosines converted to uracils in the MAGE-A1 gene after treating DNA from these cells with bisulfite?

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
-Treating DNA with the compound bisulfite converts any unmethylated cytosines into uracils. Bisulfite sequencing, which takes advantage of this mechanism, was used to compare DNA methylation of the MAGE-A1 gene in healthy cells and melanoma cells. Based on the passage, which of the following graphs most likely represents the percentage of cytosines converted to uracils in the MAGE-A1 gene after treating DNA from these cells with bisulfite?

Quizplus · Accepted Answer

11ecba2d_103e_5e10_a3bf_8ba805db95aa_MD0008_00 Gene expression is regulated in part by chromatin structure. Heterochromatin is DNA that is highly condensed and tightly wound around histones. Generally, heterochromatin exhibits low histone acetylation levels and high DNA methylation levels. Because heterochromatin is packed tightly, transcription machinery cannot access genes in these regions, leading to gene silencing. In contrast, loosely packed euchromatin is characterized by high histone acetylation levels and low DNA methylation levels. Looser euchromatin is more accessible to transcription machinery, facilitating gene expression. According to the passage, cancer/testis antigens (CTAs) are normally expressed exclusively in germline cells but are often reactivated in cancer cells. CTAs in cancer cells are often recognized as antigens (ie, foreign substances) by immune cells and elicit an immune response. Reactivation of CTAs occurs as a result of chromatin remodeling events such as DNA demethylation. Therefore, CTA genes of healthy somatic cells would likely be silenced and found in heterochromatin with high methylation levels. In contrast, CTA genes of cancer cells would likely be expressed and exist in euchromatin with low methylation levels. The question states that treating DNA with bisulfite converts any unmethylated cytosines to uracils. In this scenario, bisulfite sequencing was used to compare DNA methylation of the MAGE-A1 gene in healthy and melanoma (skin cancer) cells. The passage indicates that MAGE-A1 is likely silenced in healthy skin (somatic) cells and would therefore exhibit high numbers of methylated cytosines and low numbers of unmethylated cytosines. This would lead to a low percentage of cytosine to uracil conversions following bisulfite treatment of DNA from healthy cells. In contrast, Figure 1 shows that MAGE-A1 is expressed in melanoma cells. Accordingly, MAGE-A1 in melanoma cells is likely transcriptionally active with low numbers of methylated cytosines and high numbers of unmethylated cytosines, leading to a high percentage of cytosine to uracil conversions upon bisulfite treatment of DNA, as shown in Choice C (Choice A). (Choices B and D) The passage indicates that CTA expression differs between healthy and cancerous cells because of chromatin structure differences (eg, DNA methylation states). Accordingly, bisulfite treatment of DNA from healthy and cancerous skin cells is likely to yield contrasting (not similar) results. Educational objective: Genes found within tightly packed heterochromatin are generally silenced whereas genes found within loosely packed euchromatin are generally transcriptionally active.

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