A novel single cell PCR assay: detection of human T lymphotropic virus type I DNA in lymphocytes of patients with adult T cell leukemia

Abstract

The abnormal lymphocytes in adult T cell leukemia (ATL) reveal a peculiar morphology that is characterized by indented or lobulated nuclei. While human T lymphotropic virus type I (HTLV-I) is thought to be integrated in ATL cells, the correlation between the nuclear irregularities and HTLV-I infection is obscure. We have devised a novel single cell polymerase chain reaction (PCR) technique to examine the integration of HTLV-I provirus genome in cells from two patients with ATL. To isolate single cells, peripheral blood smears were prepared on thin polyester slides and stained with May-Grü nwald-Giemsa. Morphologically defined single cells were cut out after light microscopy. The HTLV-I DNA sequences were detected not only in ATL cells but also in normal-looking lymphocytes. This novel PCR method may provide a valuable tool for understanding the molecular events associated with HTLV-I infection at the single cell level.

Tumor suppressor genes in normal and malignant hematopoiesis

“…Furthermore, overexpression of WT-1 retarded induction of di erentiation of the HL-60 (Deuel et al, 1999) and U937 (Svedberg et al, 1998) AML cell lines and 32D cl3, a murine myeloblast cell line (Inoue et al, 1998). Also, high levels of WT-1 were detected in 68% of 600 AML and ALL samples (Miwa et al, 1992;Miyagi et al, 1993;Patmasiriwat et al, 1996;Schmid et al, 1997;Bergmann et al, 1997;Inoue et al, 1997;Im et al, 1999;Gaiger et al, 1999;Niegemann et al, 1999) and approximately 60% of MDS samples which were associated with advanced stages of the disease (Patmasiriwat et al, 1999;Tamaki et al, 1999). In summary, WT-1 is mutated in less than 10% of leukemia samples.…”

Identification of a tumor-specific methylation site in the Wilms tumor suppressor gene

“…In mice, knocking out wt1 has a striking eect on development of the mesothelium (Kreidberg et al, 1993), and malignant mesothelioma (MM) is one of a very few extrarenal neoplasms that express WT1 (Miyagi et al, 1993;Park et al, 1993;Walker et al, 1994). Development of MM in humans is closely associated with environmental or occupational exposure to asbestos or other carcinogenic mineral ®bers.…”

Application of Optical Trapping for Cells Grown on Plates: Optimization of PCR and Fidelity of DNA Sequencing of p53 Gene from a Single Cell

“…Conditions for the PK treatment before single-cell PCR vary widely in the literature, with PK at concentrations between 100 and 200 mg/L for 0.5-10 h at 40-50°C, followed by 10 -30 min at 80 -99°C to inactivate the PK (17,19,59 ). To directly test the amount of PK needed, we tested three concentrations of PK with the 5S PCR assay.…”