Recently, it was reported that exogenous bone morphogenetic protein (BMP)-2 acted as an antiproliferative agent in a variety of cell lines, including normal and cancerous gastric cell lines, indicating that BMP-2 plays an important role during cell growth. However, despite the loss of BMP-2 expression in several cancers, the underlying mechanism remains unknown. Epigenetic silencing through DNA methylation is one of the key steps during carcinogenesis. In this study, we found, through analysis by the methylation-specific polymerase chain reaction technique, CpG island methylation of the BMP-2 promoter region in gastric and colon cancer cell lines. BMP-2 mRNA was found to be activated after 5-aza-2 0 -deoxycytidine treatment of the methylation-positive cells. Moreover, 24 of the 56 (42.9%) gastric cancer tissues exhibited promoter methylation. Immunohistochemical staining revealed that 18 of the 24 (75%) gastric cancer tissues without methylation signals exhibited BMP-2 expression, whereas among 20 cancer tissues with strong methylation signals only four (20%) expressed BMP-2 (P ¼ 0.0003). These findings indicate that BMP-2 methylation is strongly associated with the loss of BMP-2 protein expression in the primary gastric carcinomas. BMP-2 methylation was more often observed in diffuse type (60.7%) than in intestinal type (25%) gastric carcinomas (P ¼ 0.007). Thus, aberrant BMP-2 methylation and the resultant loss of BMP-2 expression may be related to gastric carcinogenesis, particularly in the diffuse type.

The clinical aspects of disease progression in chronic myelogenous leukemia (CML) are well established, but the nature of the molecular events responsible is not known. We have previously reported a consistent pattern of novel sites of methylation in the 5′ region of the calcitonin (CT) gene and other chromosome 11p loci in acute myelogenous and and lymphoid leukemias. In the present study, CT gene methylation patterns were investigated in peripheral blood from 51 patients with CML. Abnormal patterns were found in only 2 of 31 patients in chronic phase, but in 5 of 8 patients in accelerated phase, and in 11 of 12 patients in blast crisis (P less than .005). For one patient studied in blast crisis, abnormal CT gene methylation was found in the peripheral blast cells but not in the granulocytes. In two of three patients studied with CML and having normal peripheral cell patterns, abnormal patterns were found in marrow blast cells. In one patient, only partial normalization of the CT gene methylation pattern was seen after chemotherapy induction of a second chronic phase and the patient relapsed 5 months later. Our findings indicate that abnormal methylation of the 5′ region of the CT gene is regularly a marker of disease progression in CML which may prove clinically useful. This abnormal methylation site is part of an imbalance in DNA methylation that may play a role in the progressive genetic instability which characterizes the advancing stages of CML.

The clinical aspects of disease progression in chronic myelogenous leukemia (CML) are well established, but the nature of the molecular events responsible is not known. We have previously reported a consistent pattern of novel sites of methylation in the 5′ region of the calcitonin (CT) gene and other chromosome 11p loci in acute myelogenous and and lymphoid leukemias. In the present study, CT gene methylation patterns were investigated in peripheral blood from 51 patients with CML. Abnormal patterns were found in only 2 of 31 patients in chronic phase, but in 5 of 8 patients in accelerated phase, and in 11 of 12 patients in blast crisis (P less than .005). For one patient studied in blast crisis, abnormal CT gene methylation was found in the peripheral blast cells but not in the granulocytes. In two of three patients studied with CML and having normal peripheral cell patterns, abnormal patterns were found in marrow blast cells. In one patient, only partial normalization of the CT gene methylation pattern was seen after chemotherapy induction of a second chronic phase and the patient relapsed 5 months later. Our findings indicate that abnormal methylation of the 5′ region of the CT gene is regularly a marker of disease progression in CML which may prove clinically useful. This abnormal methylation site is part of an imbalance in DNA methylation that may play a role in the progressive genetic instability which characterizes the advancing stages of CML.

An abnormal increase in numbers of CCGG sites methylated in the 5′ region of the human calcitonin (CT) gene occurred in tumor cell DNA samples from 90% (17 of 19) of patients with non-Hodgkin's T and B cell lymphoid neoplasms and in 95% (21 of 22) of tumor cell DNA samples from patients with acute nonlymphocytic leukemia (ANLL). The changes were not seen in patients with chronic myelogenous leukemia (0 of 9). The abnormal methylation patterns appear to be a property only of transformed or malignant cells since they were not found in DNA from nonneoplastic adult tissues including sperm, early myeloid progenitor cells, benign lymphoid hyperplasia, peripheral lymphocytes stimulated to divide, or early myeloid progenitor cells (obtained by immunoaffinity using anti-My-10 antibody), but they did appear after Epstein-Barr virus transformation of lymphocytes. Moreover, during the course of therapy in patients with ANLL, the hypermethylation pattern reflects the presence of the leukemic clone even in normal-appearing granulocytes derived from this clone. The increased methylation of the CT gene may then provide an important molecular marker for biologic events in human cell transformation or tumor progression and may prove clinically useful in monitoring patients with lymphoid and acute myelogenous neoplasms.

An abnormal increase in numbers of CCGG sites methylated in the 5′ region of the human calcitonin (CT) gene occurred in tumor cell DNA samples from 90% (17 of 19) of patients with non-Hodgkin's T and B cell lymphoid neoplasms and in 95% (21 of 22) of tumor cell DNA samples from patients with acute nonlymphocytic leukemia (ANLL). The changes were not seen in patients with chronic myelogenous leukemia (0 of 9). The abnormal methylation patterns appear to be a property only of transformed or malignant cells since they were not found in DNA from nonneoplastic adult tissues including sperm, early myeloid progenitor cells, benign lymphoid hyperplasia, peripheral lymphocytes stimulated to divide, or early myeloid progenitor cells (obtained by immunoaffinity using anti-My-10 antibody), but they did appear after Epstein-Barr virus transformation of lymphocytes. Moreover, during the course of therapy in patients with ANLL, the hypermethylation pattern reflects the presence of the leukemic clone even in normal-appearing granulocytes derived from this clone. The increased methylation of the CT gene may then provide an important molecular marker for biologic events in human cell transformation or tumor progression and may prove clinically useful in monitoring patients with lymphoid and acute myelogenous neoplasms.