The hemoregulatory peptide pEEDCK may inhibit stem cell proliferation via hydropathic binding to antisense sequence motifs in interleukin-11 and other growth factors.

In undisturbed bone marrow, most hemopoietic stem cells are nonproliferating despite the presence of multiple growth factors. Endogenous inhibitory factors are responsible for maintenance of this quiescence. Previously we sequenced and synthesized the inhibitory pentapeptide pGlu-Glu-Asp-Cys-Lys (pEEDCK), which originally derives from granulocytes, and investigated the role of this peptide in stem cell quiescence. To provide some mechanistic insight, in the present work we studied the structural relationship of this peptide to specific growth-factor-derived sequence motifs. In the murine system in vivo as well as in long-term bone marrow, antiserum to pEEDCK produced a significant stimulation of formation of colony-forming units-granulocyte/macrophage. Binding of peptides to proteins often takes place at hydropathically complementary sites. Therefore, we searched for peptides corresponding to the complementary sequence to pEEDCK. We identified antisense sequences in the genes of various cytokines and cytokine receptors including interleukin-11. The corresponding peptide Val-Leu-Leu-Thre-Arg (VLLTR) and several other peptides hydropathically complementary to pEEDCK were synthesized. We found that pEEDCK binds specifically to these peptides as well as to complete interleukin-11. Dissociation constants were in the 10 microM range. The peptide hydropathically corresponding to pEEDCK (VLLTR) was found to stimulate colony-forming units-granulocyte/macrophage formation. Our data suggest that pEEDCK could exert a coordinating function in the hemopoietic cytokine network by binding to multiple regulatory proteins and modulating their activity.

Analysis of peripheral blood progenitor cells demonstrates limitations of minimal residual disease diagnosis in a case of acute promyelocytic leukemia.

Detection of minimal residual disease (MRD) by analysis of the PML-RAR alpha fusion transcript using the RT-PCR method is routinely carried out on peripheral blood and bone marrow of patients with APL (AML, FAB:M3). Therapy aims to achieve repeated negative results in these patients thus confirming clinical complete remission. We report a case of APL in second complete remission in which no leukemic cells had been detected in BM and PB for 20 months, and in which PBPC-pheresis was carried out for future transplantation. In two of five pheresis PML-RAR alpha fusion transcripts were detected. This shows that the residual leukemic population may only reach detection level after enrichment by PBPC-pheresis.

Chromosomal insertion of human papillomavirus 18 sequences in HeLa cells detected by nonisotopic in situ hybridization and reflection contrast microscopy.

Genomic insertion of human papillomavirus (HPV) sequences is associated with the genesis of cervical carcinoma, and HPV-induced incipient cellular alterations may also present a requisite for the establishment of cell lines such as HeLa. Considering the theoretical importance of specific viral integration sites, we attempted to detect in HeLa cells the chromosomal location of DNA sequences homologous to HPV-16 and HPV-18 sequences by a nonisotopic high resolution in situ hybridization technique. Chromosome identification following in situ hybridization was possible by counterstaining of the same preparation with Chromomycin A3, Distamycin A, and DAPI. Using this approach, we have assigned HPV-18 integration in HeLa cells to band 8q24 (a site including the locus of the myc-protooncogene), to an abnormal chromosome 22, and to a not yet identified marker chromosome possibly neighboring other oncogenic or activating sites. The sensitive detection technique described in this study presents a new approach involving in situ chromosome hybridization with biotinylated DNA probes in combination with reflection contrast microscopy and subsequent fluorescent R- and C-banding. The method allowed the assignment of a 7-kb HPV-18 DNA probe to human chromosomal sites important in growth regulation and cancerogenesis. It should prove useful in a number of similar studies using other viral and oncogenic DNA probes.