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Aberrant allele-specific replication, independent of parental origin, in blood cells of cancer patients.

Dotan ZA, Dotan A, Ramon J, Avivi L - BMC Cancer (2008)

Bottom Line: Malignancy was documented to be associated with gross modifications in the inherent replication-timing coordination between allelic counterparts of imprinted genes as well as of biallelically expressed loci.The non-disease specific aberrant epigenetic profile displayed in peripheral blood cells of patients with a solid tumour (unlike genetic aberrations) can be reversed, by an epigenetic drug applied in vitro, to the normal.It appears that the cancerous status differentiates between two allelic counterparts in a non-random manner, but independent of the parental origin.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Urology, Sheba Medical Center, Tel-Hashomer 52621, Israel. Zohar.Dotan@sheba.health.gov.il

ABSTRACT

Background: Allelic counterparts of biallelically expressed genes display an epigenetic symmetry normally manifested by synchronous replication, different from genes subjected to monoallelic expression, which normally are characterized by an asynchronous mode of replication (well exemplified by the SNRPN imprinted locus). Malignancy was documented to be associated with gross modifications in the inherent replication-timing coordination between allelic counterparts of imprinted genes as well as of biallelically expressed loci. The cancer-related allelic replication timing aberrations are non-disease specific and appear in peripheral blood cells of cancer patients, including those with solid tumors. As such they offer potential blood markers for non-invasive cancer test. The present study was aimed to gain some insight into the mechanism leading to the replication timing alterations of genes in blood lymphocytes of cancer patients.

Methods: Peripheral blood samples derived from patients with prostate cancer were chosen to represent the cancerous status, and samples taken from patients with no cancer but with benign prostate hyperplasia were used to portray the normal status. Fluorescence In Situ Hybridization (FISH) replication assay, applied to phytohemagglutinin (PHA)-stimulated blood lymphocytes, was used to evaluate the temporal order (either synchronous or asynchronous) of genes in the patients' cells.

Results: We demonstrated that: (i) the aberrant epigenetic profile, as delineated by the cancer status, is a reversible modification, evidenced by our ability to restore the normal patterns of replication in three unrelated loci (CEN15, SNRPN and RB1) by introducing an archetypical demethylating agent, 5-azacytidine; (ii) following the rehabilitating effect of demethylation, an imprinted gene (SNRPN) retains its original parental imprint; and (iii) the choice of an allele between early or late replication in the aberrant asynchronous replication, delineated by the cancer status, is not random but is independent of the parental origin.

Conclusion: The non-disease specific aberrant epigenetic profile displayed in peripheral blood cells of patients with a solid tumour (unlike genetic aberrations) can be reversed, by an epigenetic drug applied in vitro, to the normal. It appears that the cancerous status differentiates between two allelic counterparts in a non-random manner, but independent of the parental origin.

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Characterization of the SD-cell populations for SNRPN and CEN15 in cell samples of individuals heterozygous for the CEN15 size, showing one large and one small CEN15 marker. Light bars show the portion (%) of the relevant SD-cell population in which the replicated region (D-signal) is associated with the large CEN15 marker, and dark bars show the SD-cell portion in which the D-signal is associated with the small CEN15 marker. (a) and (b) samples of cases free of cancer (K2 was from a healthy young man, the son of K); (c) and (d) samples of cancer patients; the last bar in frame (c) is a sample of cancer patient C (case C5 in Fig. 3) grown in the presence of AZA; at least 200 SD cells were scored from each sample for each assay.
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Figure 4: Characterization of the SD-cell populations for SNRPN and CEN15 in cell samples of individuals heterozygous for the CEN15 size, showing one large and one small CEN15 marker. Light bars show the portion (%) of the relevant SD-cell population in which the replicated region (D-signal) is associated with the large CEN15 marker, and dark bars show the SD-cell portion in which the D-signal is associated with the small CEN15 marker. (a) and (b) samples of cases free of cancer (K2 was from a healthy young man, the son of K); (c) and (d) samples of cancer patients; the last bar in frame (c) is a sample of cancer patient C (case C5 in Fig. 3) grown in the presence of AZA; at least 200 SD cells were scored from each sample for each assay.

Mentions: Using two-color FISH to identify the CEN15 and the SNRPN in the same cell, we examined the population of SD cells for SNRPN in the samples heterozygous for the large CEN15 marker. The early replicating SNRPN allele was not randomly distributed between the two chromosome-15s in either the informative (heterozygous) samples derived from non-cancer cases (Fig. 4a) or those derived from the informative cancer cases (Fig. 4c). Specifically, in the samples of the non-cancer BPH cases (G, R and K), in the K2 sample, and in one sample of a cancer (CAP) case (J), the early replicating SNRPN allele was located in more than 70% of SD cells on the chromosome carrying a large CEN15 marker. On the other hand, in samples V and Z derived from non-cancer patients, as well as samples Y, A, and C derived from cancer patients, the early replicating SNRPN allele was found in more than 75% of SD cells on the chromosome identified by a small CEN15 marker (Fig. 4a and 4c). This clearly showed that the relaxation in the asynchronous replication of SNRPN (reduced SD-cell frequency) that characterizes samples of cancer patients was not accompanied by randomization of the early and late replicating alleles.


Aberrant allele-specific replication, independent of parental origin, in blood cells of cancer patients.

Dotan ZA, Dotan A, Ramon J, Avivi L - BMC Cancer (2008)

Characterization of the SD-cell populations for SNRPN and CEN15 in cell samples of individuals heterozygous for the CEN15 size, showing one large and one small CEN15 marker. Light bars show the portion (%) of the relevant SD-cell population in which the replicated region (D-signal) is associated with the large CEN15 marker, and dark bars show the SD-cell portion in which the D-signal is associated with the small CEN15 marker. (a) and (b) samples of cases free of cancer (K2 was from a healthy young man, the son of K); (c) and (d) samples of cancer patients; the last bar in frame (c) is a sample of cancer patient C (case C5 in Fig. 3) grown in the presence of AZA; at least 200 SD cells were scored from each sample for each assay.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2629776&req=5

Figure 4: Characterization of the SD-cell populations for SNRPN and CEN15 in cell samples of individuals heterozygous for the CEN15 size, showing one large and one small CEN15 marker. Light bars show the portion (%) of the relevant SD-cell population in which the replicated region (D-signal) is associated with the large CEN15 marker, and dark bars show the SD-cell portion in which the D-signal is associated with the small CEN15 marker. (a) and (b) samples of cases free of cancer (K2 was from a healthy young man, the son of K); (c) and (d) samples of cancer patients; the last bar in frame (c) is a sample of cancer patient C (case C5 in Fig. 3) grown in the presence of AZA; at least 200 SD cells were scored from each sample for each assay.
Mentions: Using two-color FISH to identify the CEN15 and the SNRPN in the same cell, we examined the population of SD cells for SNRPN in the samples heterozygous for the large CEN15 marker. The early replicating SNRPN allele was not randomly distributed between the two chromosome-15s in either the informative (heterozygous) samples derived from non-cancer cases (Fig. 4a) or those derived from the informative cancer cases (Fig. 4c). Specifically, in the samples of the non-cancer BPH cases (G, R and K), in the K2 sample, and in one sample of a cancer (CAP) case (J), the early replicating SNRPN allele was located in more than 70% of SD cells on the chromosome carrying a large CEN15 marker. On the other hand, in samples V and Z derived from non-cancer patients, as well as samples Y, A, and C derived from cancer patients, the early replicating SNRPN allele was found in more than 75% of SD cells on the chromosome identified by a small CEN15 marker (Fig. 4a and 4c). This clearly showed that the relaxation in the asynchronous replication of SNRPN (reduced SD-cell frequency) that characterizes samples of cancer patients was not accompanied by randomization of the early and late replicating alleles.

Bottom Line: Malignancy was documented to be associated with gross modifications in the inherent replication-timing coordination between allelic counterparts of imprinted genes as well as of biallelically expressed loci.The non-disease specific aberrant epigenetic profile displayed in peripheral blood cells of patients with a solid tumour (unlike genetic aberrations) can be reversed, by an epigenetic drug applied in vitro, to the normal.It appears that the cancerous status differentiates between two allelic counterparts in a non-random manner, but independent of the parental origin.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Urology, Sheba Medical Center, Tel-Hashomer 52621, Israel. Zohar.Dotan@sheba.health.gov.il

ABSTRACT

Background: Allelic counterparts of biallelically expressed genes display an epigenetic symmetry normally manifested by synchronous replication, different from genes subjected to monoallelic expression, which normally are characterized by an asynchronous mode of replication (well exemplified by the SNRPN imprinted locus). Malignancy was documented to be associated with gross modifications in the inherent replication-timing coordination between allelic counterparts of imprinted genes as well as of biallelically expressed loci. The cancer-related allelic replication timing aberrations are non-disease specific and appear in peripheral blood cells of cancer patients, including those with solid tumors. As such they offer potential blood markers for non-invasive cancer test. The present study was aimed to gain some insight into the mechanism leading to the replication timing alterations of genes in blood lymphocytes of cancer patients.

Methods: Peripheral blood samples derived from patients with prostate cancer were chosen to represent the cancerous status, and samples taken from patients with no cancer but with benign prostate hyperplasia were used to portray the normal status. Fluorescence In Situ Hybridization (FISH) replication assay, applied to phytohemagglutinin (PHA)-stimulated blood lymphocytes, was used to evaluate the temporal order (either synchronous or asynchronous) of genes in the patients' cells.

Results: We demonstrated that: (i) the aberrant epigenetic profile, as delineated by the cancer status, is a reversible modification, evidenced by our ability to restore the normal patterns of replication in three unrelated loci (CEN15, SNRPN and RB1) by introducing an archetypical demethylating agent, 5-azacytidine; (ii) following the rehabilitating effect of demethylation, an imprinted gene (SNRPN) retains its original parental imprint; and (iii) the choice of an allele between early or late replication in the aberrant asynchronous replication, delineated by the cancer status, is not random but is independent of the parental origin.

Conclusion: The non-disease specific aberrant epigenetic profile displayed in peripheral blood cells of patients with a solid tumour (unlike genetic aberrations) can be reversed, by an epigenetic drug applied in vitro, to the normal. It appears that the cancerous status differentiates between two allelic counterparts in a non-random manner, but independent of the parental origin.

Show MeSH
Related in: MedlinePlus