Philadelphia chromosome-positive cells are equally distributed in AC133+ and AC133− fractions of CD34+ peripheral blood progenitor cells from patients with CML
1466 negative for CD14 (DAKO, Glostrup, Denmark), which is a marker of macrophages, indicating that dendritic cells were contained in the stromal cell layer. For first PCR reaction 100 ng of genomic DNA was utilized. A primer pair was designed from KSHV genomic DNA sequences (GenBank accession number; U18551) as follows: forward; 59-TGTATTAGCTAACCCTTCTAGCG-39, reverse; 59-TCCGTG TTGTCTACGTCCAG-39. Using this primer pair, a 620 bp DNA fragment that contained the KS330233 sequence was amplified. We further carried out nested PCR using two sets of primers (Figure 1, upper panel). Primers for nested PCR are as follows: S1; 59CAGACTCTTCGCTGATGAAC-39, S2; 59-TCATGGCACTCGACAAGAGT-39, A1; 59-AGATGTGGTACACCAACAGC-39, A2; 59-CCCATAAATGACACATTGGTGG-39. This system allowed us to detect KSHV genome from at least 1 pg of genomic DNA from BCBL-1, a Kaposi’s sarcoma-derived cell line which is known to be infected by KSHV (kindly supplied by Drs Michael McGrath and Don Ganem through the AIDS Research and Reference Reagent Program, Division of AIDS, NIADA, NIH, Bethesda, MD, USA). A b-globin genomic sequence was amplified as an internal control using a primer pair purchased from TaKaRa Biomedicals (Tokyo, Japan). Results are shown in the lower panel of Figure 1. In all samples, including all stromal cell samples, no KSHV sequence was detected by nested-PCR, although a clear band was detected in the positive control. The present results indicated no correlation between the occurrence of MM and KSHV infection and did not corroborate the report by Rettig et al. Rettig et al6 claimed that heparin contamination in the sample may decrease the sensitivity of PCR. However, since we utilized frozen cells or long-term cultured stromal cells as sources of DNA, heparin contamination should be negligible. Rettig et al1 also reported that KSHV was found only in stromal cells but not in myeloma cells. However, if stromal cells were infected with KSHV, nested-PCR analysis should be sensitive enough to detect the KSHV genome. Moreover, there was no KSHV DNA detected in purified stromal cells when four cases were examined. Our results suggest that KSHV does not play a significant role in the pathogenesis of MM in the Japanese population.