LacZ staining in paraffin-embedded tissue sections.

Femora and tibiae of rats carrying leukemia from a LacZ-marked acute promyelocytic leukemia-derived leukemic cell line (LT12NL15) were decalcified using EDTA and routinely embedded in paraffin. Sections were used to develop for the first time an immunostaining method for LacZ, employing catalyzed reporter deposition (CARD) based on the deposition of biotinylated tyramine. This method was used to study homing and adhesion of leukemic cells.

PCR amplification of short tandem repeat sequences allows serial studies of chimaerism/engraftment following BMT in rodents.

Animal models of bone marrow transplantation (BMT) allow evaluation of new experimental treatment strategies. One potential strategy involves the treatment of donor marrow with ultra-violet B light to allow transplantation across histocompatibility boundaries without an increase in graft rejection or graft-versus-host disease. A major requirement for a new experimental protocol, particularly if it involves manipulation of the donor marrow, is that the manipulated marrow gives rise to long-term multilineage engraftment. DNA based methodologies are now routinely used by many centres to evaluate engraftment and degree of chimaerism post-BMT in humans. We report the adaptation of this methodology to the serial study of engraftment in rodents. Conditions have been defined which allow analysis of serial tail vein samples using PCR of short tandem repeat sequences (STR-PCR). These markers have been used to evaluate the contribution of ultraviolet B treated marrow to engraftment following BMT in rodents without compromising the health of the animals under study. Chimaerism data from sequential tail vein samples and bone marrow from selected sacrificed animals showed excellent correlation, thus confirming the validity of this approach in analysing haemopoietic tissue. Thus the use of this assay may facilitate experimental studies in animal BMT.

Homing of fluorescently labeled murine hematopoietic stem cells.

PKH-26 was used as a viable fluorescent membrane stain for murine hematopoietic stem cells. The presence of the dye on the cells was shown not to interfere with their ability to form day-8 and -12 spleen colonies in lethally irradiated mice. To study their in vivo homing behavior in detail, 10(4) labeled cells from a population enriched for CFU-S were injected intravenously into nonirradiated mice and into mice irradiated 3 hours previously. At 17, 41, and 65 hours after injection, the numbers of labeled cells per organ were quantified using the specialty developed flow cytometric fluorescence hypercompensation procedure for the detection of rare events, which allows a detection sensitivity of 1 per 10(6). Spleen homing in irradiated and nonirradiated mice was virtually identical, whereas homing to nonirradiated bone marrow was 2.5 times higher than to irradiated bone marrow. This indicates a different homing mechanism for spleen and bone marrow. The results of this direct homing assay were placed in perspective with results of indirect homing studies from the literature, introducing a new "h-factor." From the CFU-S data, putative specific enrichment factors for spleen-specific and bone marrow-specific homing were derived. Examination of the fluorescence intensity distribution among the labeled cell population indicated that virtually all cells started to proliferate rapidly after injection into both irradiated and nonirradiated animals. This indicates that specific signals from stromal elements in the stem cell niches are needed to keep the cells quiescent and that the majority of the transplanted stem cells do not home to such niches. The potential use of PKH-26 for in vivo characterization of stem cell niches is discussed.

Monitoring of leukemia growth in a rat model using a highly sensitive assay for the detection of LacZ marked leukemic cells.

A very sensitive assay for the detection of LacZ marked cells of an in vitro growing subline of the brown Norway rat myelocytic leukemia (BNML) model was developed. By combining cytochemical X-gal staining with D-galactose mediated suppression of endogenous background beta-galactose activity, a detection sensitivity of one leukemic cell per 10(8) normal bone marrow cells could be achieved. A detailed analysis of the in vivo growth pattern and kinetics of this cell line is presented. Also, it is shown that after cyclophosphamide treatment of leukemic rats no leukemic colonies are formed in an agar-colony assay, whereas the leukemic cells remain detectable in the bone marrow for a considerable time period. Eventually, however, all leukemic cells disappear from the marrow. These findings are discussed in the light of prolonged detection of rare leukemic cells in patients in continuing remission.

Comparison of the response to T-cell activation by integrated HIV-1 and HTLV-1 LTR-lacZ vectors.

Human Jurkat T-cell clones containing stably integrated HIV-1 LTR or HTLV-1 LTR/lacZ vectors were studied to compare the responses of integrated LTRs to T-cell activation. Responses were compared also with those obtained in parallel with Jurkat cells stably expressing lacZ under the control of the cellular enhancer element NF-AT of the IL-2 promoter. Activation induced via the cell surface TCR/CD3 complex or the CD28 receptor elicited responses from the LTR of HIV-1; however, HTLV-1 LTR-directed expression was not observed following triggering of these cell surface pathways. Mitogenic activation by elevation of intracellular calcium (Ca2+) levels along with protein kinase C (PKC) signals was required for optimal expression of the HIV-1 LTR and the NF-AT element; however, increased intracellular Ca2+ was inhibitory to PKC-mediated expression from the HTLV-1 LTR. Time course experiments revealed a sustained PKC-mediated response by the HTLV-1 LTR, which was detectable in the absence of Ca2+ as early as 6 hr following stimulation. In contrast to the HTLV-1 LTR, in time course experiments the HIV-1 LTR responded to stimulation by mitogenic activation of PKC in the absence and presence of Ca2+ and by antiCD3 with lacZ expression beginning as early as 3 hr poststimulation. These results suggest that the HTLV-1 LTR appears to be refractory to several cellular pathways which are upregulatory to the HIV-1 LTR.

Differential suppression of background mammalian lysosomal beta-galactosidase increases the detection sensitivity of LacZ-marked leukemic cells.

A method is described for the detection of Escherichia coli beta-galactosidase-expressing leukemic cells in ex vivo bone marrow samples. 4-Methylumbelliferyl-beta-D-galactopyranoside is used as a substrate in a kinetic assay. D-Galactose is used to suppress endogenous lysosomal beta-galactosidase activity, yielding a sixfold increase in sensitivity. With this assay, the detection limit is one leukemic cell per 10(4) normal bone marrow cells.

Retrovirus-mediated transfer and expression of marker genes in the BN rat acute myelocytic leukemia model for the study of minimal residual disease (MRD).

To study minimal residual disease (MRD) in leukemia, we transferred the Escherichia coli genes encoding beta-galactosidase (lacZ) and neomycin resistance (neo(r)) into the subline LT12 of the Brown Norway rat acute myelocytic leukemia (BNML), employing the retroviral BAG vector. In this way leukemic cells were genetically marked. Ten independent cell lines were characterized during in vitro growth as well as during two subsequent in vivo passages for expression of neo(r) for which the neomycin analogue G418 was used, and for lacZ expression for which the substrate 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal) was used. Out of 10 lines, four revealed permanent high expression of lacZ in all cells. In four other lines greatly varying lacZ expression between the individual cells from these lines was observed. In the remaining two lines lacZ expression was gradually lost. In contrast, neo(r) expression was gradually lost in eight out of the 10 lines, particularly rapidly during in vivo passaging. In the remaining two lines neo(r) expression was retained. The genetic modification did not alter the in vitro leukemogenicity of the cells. Long term in vivo expression of neo(r) and lacZ was followed in two selected lines up to 12 subsequent passages, i.e. one from the group of homogeneous high lacZ expression and one from the group of heterogeneous lacZ expression. In both lines lacZ expression was retained whereas neo(r) expression was rapidly lost after the third passage. The feasibility of using genetically marked leukemic cells for studies of minimal residual disease (MRD) was explored by injecting rats with leukemic cells, treating them with chemotherapy at full blown leukemia development to reduce the tumor load, mimicking the induction of a state of MRD and studying lacZ expression at relapse. LacZ expression was evident in 100% of the cells whereas neo(r) expression was lost in a considerable fraction. These results indicate that the viral vector BAG can be used to mark leukemia cells genetically although a selection of clones with the desired stability of long-term expression is required.

Construction and expression of an adenosine deaminase::lacZ fusion gene.

A eukaryotic expression vector was constructed in which the coding nucleotide sequences (ADA) of human adenosine deaminase (ADA) were fused in frame with the coding sequences of the bacterial gene lacZ encoding beta-galactosidase (beta Gal). This ADA::lacZ fusion gene was anticipated to encode a hybrid protein that has retained the biological functions of both proteins. Transfection of mammalian cells with the fusion gene resulted in the synthesis of both ADA and beta Gal. Cells expressing the gene could therefore be detected with the histochemical staining procedure that relies on the conversion of the indicator, XGal, by beta Gal. In addition, the transfected cells could be sorted on a fluorescence-activated cell sorter with the use of a vital staining procedure described for the selection of beta Gal-producing cells. Cell lines that harbored the fusion gene were tested for ADA overexpression by exposing them to the cytotoxic adenosine analog 9-beta-D-xylofuranosyl adenine (Xyl-A), in the presence of the ADA inhibitor deoxycoformycin (dCF). Resistance to Xyl-A/dCF was observed in the lines carrying ADA::lacZ and moreover, the fraction of cells that survived a stringent selection for ADA overexpression also exhibited significantly increased levels of beta Gal, which confirmed the direct linkage between ADA and lacZ expression. The use of this and other fusion genes might be useful in the development of gene-therapy protocols where they could help to meet the demand for versatile methods to detect and select cells with newly introduced genes.