Monoclonal antibody BB9 raised against bone marrow stromal cells identifies a cell-surface glycoprotein expressed by primitive human hemopoietic progenitors.

OBJECTIVE: The identification of cell-surface antigens whose expression is limited to primitive hematopoietic progenitor cells (HPC) is of major value in the identification, isolation, and characterization of candidate stem cells in human hemopoietic tissues. Based on the observation that bone marrow stromal cells and primitive HPC share several cell-surface antigens, we sought to generate monoclonal antibodies to HPC by immunization with cultured human stromal cells. METHODS: BALB/c mouse were immunized with human bone marrow (BM)-derived stromal cells. Splenocytes isolated from immunized mice were fused with the NS-1 murine myeloma cell line and resulting hybridomas selected in HAT medium, then screened for reactivity against stromal cells, peripheral blood (PB), and BM cells. RESULTS: A monoclonal antibody (MAb), BB9, was identified based on its binding to stromal cells, a minor subpopulation of mononuclear cells in adult human BM, and corresponding lack of reactivity with leukocytes in PB. BB9 bound to a minor subpopulation of BM CD34(+) cells characterized by high-level CD34 antigen and Thy-1 expression, low-absent expression of CD38, low retention of Rhodamine 123, and quiescent cycle status as evidenced by lack of labeling with Ki67. CD34(+)BB9(+) cells, in contrast to CD34(+)BB9(-) cells, demonstrated a capacity to sustain hematopoiesis in pre-CFU culture stimulated by the combination of IL-3, IL-6, G-CSF, and SCF. BB9 also demonstrated binding to CD34(+) cells from mobilized PB. CONCLUSION: Collectively, these data therefore demonstrate that MAb BB9 identifies an antigen, which is selectively expressed by hierarchically primitive human HPC and also by stromal cells.

Combination of stem cell factor and granulocyte colony-stimulating factor mobilizes the highest number of primitive haemopoietic progenitors as shown by pre-colony-forming unit (pre-CFU) assay.

Fifty-two patients with poor prognosis carcinoma of the breast underwent peripheral blood stem cell (PBSC) mobilization using five different regimens. The yields of primitive haemopoietic progenitors were quantified by a recently described pre-colony-forming unit (pre-CFU) assay using limiting dilution analysis (LDA). Results of days 14 and 35 pre-CFU were also correlated with conventional CD34+ cell enumeration, CFU-GM (granulocyte-macrophage) and long-term culture-initiating cell (LTCIC) assays. The yield of pre-CFUs with the combination of granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF) was significantly higher than with G-CSF alone, cyclophosphamide (Cyclo) and granulocyte-monocyte colony-stimulating factor (GM-CSF), interleukin (IL)-3 and GM-CSF, or Cyclo alone. No significant correlation between neutrophil engraftment and pre-CFU could be demonstrated. Furthermore, CFU-GM was shown to bear a stronger correlation with pre-CFU and LTCIC than CD34+ cell measurement; thus, CFU-GM remains a useful biological tool for haemopoietic stem cell assay. We conclude that the combination of G-CSF and SCF mobilizes the highest number of pre-CFUs as measured by functional pre-CFU assay, which provides an alternative measurement of primitive haemopoietic progenitors to the LTCIC assay.

Increased recruitment of hematopoietic progenitor cells underlies the ex vivo expansion potential of FLT3 ligand.

The ligand for flt-3 (FLT3L) exhibits striking structural homology with stem cell factor (SCF) and monocyte colony-stimulating factor (M-CSF) and also acts in synergy with a range of other hematopoietic growth factors (HGF). In this study, we show that FLT3L responsive hematopoietic progenitor cells (HPC) are CD34+CD38-, rhodamine 123dull, and hydroperoxycyclophosphamide (4-HC) resistant. To investigate the basis for the capacity of FLT3L to augment the de novo generation of myeloid progenitors from CD34+CD38- cells, single bone marrow CD34+CD38- cells were sorted into Terasaki wells containing serum-free medium supplemented with interleukin-3 (IL-3), IL-6, granulocyte colony-stimulating factor (G-CSF), SCF (4 HGF) +/- FLT3L. Under these conditions, FLT3L recruited approximately twofold more CD34+CD38- cells into division than 4 HGF alone. The enhanced proliferative response to FLT3L was evident by day 3 and was maintained at all subsequent time points examined. In accord with these findings, we also show that transduction of CD34+CD38- cells with the LAPSN retrovirus is enhanced by FLT3L. The results of these experiments therefore indicate that increased recruitment of primitive HPC into cell cycle underlies the ex vivo expansion potential of FLT3L and also its ability to improve retroviral transduction of HPC.

Mutagenic properties of the T-C cyclobutane dimer.

G x C-->A x T transitions within T-C or C-C bipyrimidine sequences are by far the most frequent class of mutation induced by 254-nm UV irradiation in most genes and species investigated, but the reason for the high degree of mutability and specificity at these sites is uncertain. Some data implicate the deamination of cytosine to uracil as a possible cause, but other results appear to indicate that the rate of deamination is too low for this to be significant in Escherichia coli. If deamination is not the cause, the high degree of mutability must presumably reflect the inherent properties of T-C and C-C dimers. We investigated this question by transfecting excision-deficient and excision-proficient strains of E. coli with single-stranded vectors that carried a site-specific cis-syn T-C cyclobutane dimer and by analyzing the nucleotide sequences of replicated vector products. We found that replication past the T-C dimer, like replication past its T-T and U-U counterparts, is in fact >95% accurate and that the frequencies of bypass are also very similar for these photoproducts. Since the T-C dimer appears to be only weakly mutagenic, the high frequency of UV-induced mutations at T-C sites presumably depends on some other process, such as deamination, although the mechanism remains to be established.

Accuracy of replication past the T-C (6-4) adduct.

The thymine-cytosine pyrimidine-pyrimidone (6-4) adduct has variously been predicted to be among the most and among the least mutagenic of the ultraviolet light photoproducts. We have therefore investigated the frequency and accuracy of DNA replication past this lesion, using a single-stranded M13mp7-based vector with a uniquely located example of this lesion transfected into SOS-induced and uninduced cells of a uvr A6 strain of Escherichia coli. Both the UVC T-C (6-4) adduct and its Dewar valence (UVB) photoisomer were studied. Random samples from non-selective collections of progeny phage were sequenced to determine the nature of the replication events that occurred at or near the site of template damage under SOS conditions. The UVC (6-4) adduct was found to be much less mutagenic than its T-T counterpart, but still much more mutagenic than a cyclobutane dimer; 34% (71 out of 206) of all bypass events yielded mutations, of which all were targeted and 80% (57 out of 71) were 3' C-->T transitions. The Dewar valence photoisomer exhibited reduced specificity and enhanced mutagenicity; 79% (183 out of 233) of the phage progeny were mutants, of which all but one were targeted and 45% (83 out of 183) were 3' C-->T transitions. For the most part, these results are consistent with a model postulating base-pairing between the pyrimidinone (of either the C or T variety) and guanine, via hydrogen bonds at N-3 and O-2 in the UVC, but not the Dewar, isomer. The occurrence of the 3' C-->T transitions, not predicted by this model, shows however that the absence of a methyl group at C-5 also has a significant influence on mutation induction. Both isomers were efficient blocks to replication; less than 1% of these vectors could be replicated in uninduced cells. Following SOS induction the frequency of bypass increased to 24.5% and 12.5% for the UVC and the Dewar isomers, respectively.

Mutagenesis induced by single UV photoproducts in E. coli and yeast.

Data from experiments with single-stranded vectors that carry a site-specific cyclobutane dimer, pyrimidine (6-4) pyrimidone adduct, or abasic lesion, replicated in either E. coli or, in some cases, bakers' yeast, Saccharomyces cerevisiae, are used to examine two questions: (i) what factors are responsible for the lesion's mutagenicity? and (ii) what are the relative contributions of different photoproducts to the spectrum of UV-induced mutations? With respect to the first question, we suggest that the structure of the mutagen-modified template itself largely determines the kinds of mutations induced, but the relative frequencies of these mutations, the error frequency, and the bypass frequency are strongly dependent on the particular organism studied. With respect to the second question, we suggest that cyclobutane dimers may be responsible for most of the mutations in slowly replicating genomes because of the deamination of cytosine, and that the T-T, and to a lesser extent the T-C, (6-4) adducts play a greater role in the UV mutagenesis of quickly replicating viruses, such as M13 and lambda phage.

Specificity of SOS mutagenesis in native M13lacI phage.

Base substitutions account for 90% of all forward mutations sequenced in unmodified M13lacI DNA grown in both UV-irradiated and nonirradiated hosts. The principal effect of SOS induction was an increase in the contribution of transversions, in particular A.T----T.A events.

Spontaneous and 9-aminoacridine-induced frameshift mutagenesis: second-site frameshift mutation within the N-terminal region of the lacI gene of Escherichia coli.

A novel forward mutational system, based on the acquisition of an Iq-d dominant phenotype from an initial Iq- recessive state, was used to identify second-site frameshift mutation [+/- 1(+/- 3n) events] within the N-terminal region of the lacI gene of Escherichia coli. The DNA sequences are described of forty-six spontaneous and twenty 9-aminoacridine(9-AA)-induced second site mutations. Although -1 frameshift events dominate both spectra, the nature and site specificity of these events clearly distinguish two mutational distributions. The spontaneous distribution contains two -(A:T) frameshift hotspots; one within a monotonic A5 run (9 occurrences), the other at a 5'-CACAACAAC-3' sequence (12 occurrences). In contrast 17 of the 20 mutations recovered after 9-AA treatment involve the loss of a G:C pair, 14 of which occur at a single site (5'-CGGGC-3'). The striking specificity of the observed mutational hotspots is of interest since this open genetic target contains similar sequences which were infrequently recovered.

Mutational specificities of environmental carcinogens in the lacl gene of Escherichia coli H. V: DNA sequence analysis of mutations in bacteria recovered from the liver of Swiss mice exposed to 1,2-dimethylhydrazine, azoxymethane, and methylazoxymethanolacetate.

The host-mediated assay (HMA) was used to determine the spectra of mutations induced in the lacl gene of Escherichia coli cells recovered from the livers of Swiss mice exposed to the carcinogens 1,2-dimethylhydrazine (SDMH), azoxymethane (AOM), and methylazoxymethanolacetate (MAMA). These spectra were further compared with changes induced by dimethylnitrosamine (DMNA) in the HMA methodology. A total of 177 independent lacl mutations arising in the HMA following exposure to SDMH, AOM, and MAMA were analyzed. Single-base substitutions accounted for 97% of all mutations analyzed. The vast majority of the single-base substitutions consisted of G:C----A:T transitions (94% of all mutations). The remaining mutations consisted of A:T----G:C transitions (3% of all mutations) while non-base substitutions accounted for only 3% of the total mutagenesis. The latter mutations consisted of one frameshift mutation and four lacO deletions. The distribution of G:C----A:T transitions induced by the three chemicals in the first 200 bp of the lacl gene was not random, but rather clustered at sites where a target guanine was flanked at the 5' site by a purine residue.

Mutational specificity of alkylating agents and the influence of DNA repair.

Alkylating treatments predominantly induce G: C = greater than A:T transitions, consistent with the predicted significance of the miscoding potential of the O6-alG lesion. However, the frequency and distribution of these events induced by any one compound may be diagnostic. SN1 agents that act via an alkyldiazonium cation, such as the N-nitroso compounds, preferentially generate G: C = greater than A:T transitions at 5'-RG-3' sites, while the more SN2 alkylsulfates and alkylalkane-sulfonates do not. The precise nature of this site bias and the possibility of strand bias are target dependent. The extent of this site bias and the contribution of other base substitutions are substituent size dependent. A similar 5'-RT-3' effect is seen for A:T = greater than G:C transitions, presumably directed by O4-alT lesions. The 5'-RG-3' effect, at least, likely reflects a deposition specificity arising from some aspect of helix geometry, although it may be further exaggerated by alkylation-specific repair. Excision repair appears to preferentially reduce the occurrence of ethylation-induced G:C = greater than A:T and A:T = greater than G:C transitions at sites flanked by A:T base pairs. This may be due to an enhancement of the helical distortion imposed by damage at such positions. A similar effect is not seen for methylation-induced mutations and in the case of propyl adducts, the influence of excision repair on the ultimate distribution of mutation cannot be as easily defined with respect to neighbouring sequence.

The dissimilar mutational consequences of SN1 and SN2 DNA alkylation pathways: clues from the mutational specificity of dimethylsulphate in the lacI gene of Escherichia coli.

The mutational specificity of the monofunctional alkylating agent dimethylsulphate has been determined through the DNA sequence characterization of 121 lacI-d mutations of Escherichia coli. The predominant mutation induced was the G:C----A:T transition (75%). Transversions constituted 20% of all mutation with the greatest contribution being that of G:C----T:A events (12%). Runs of G:C base pairs were the preferred sites of frameshift mutation. One 6-bp sequence (5'-CCCGCG-3') appeared to be highly susceptible to all classes of mutation and events within this sequence accounted for 33% of all mutations characterized. Although the distribution of G:C----A:T mutations appeared non-random, the site-specificity observed was quite different from that reported for SN1 alkylating agents. The results of this study highlight the differences between the consequences of SN1 and SN2 alkylation pathways.

Mutational specificities of environmental carcinogens in the lacI gene of Escherichia coli. I. The direct-acting analogue N-nitroso-N-methyl-N-alpha-acetoxymethylamine.

The mutational specificity of N-nitroso-N-methyl-N-alpha-acetoxymethylamine in Escherichia coli has been determined through the DNA sequence characterization of 190 forward mutations in the lacI gene. Consistent with the methylating ability of this compound and the predicted mutagenic potential of O6-methylguanine damage, the predominant mutation was the G:C----A:T transition. An analysis of the neighbouring template revealed a similar 5' flanking sequence influence on G:C----A:T site mutability reported for other direct-acting SN1 alkylating agents. However, a dose-dependency was observed. The preference for transition at guanine residues flanked (5') by a purine over those preceded by a pyrimidine decreased from a ratio of 19:1 (upon 1 mM treatment) to 6:1 (upon 10 mM treatment) and 4:1 (upon 30 mM treatment). Two double G:C----A:T transition mutants were characterized. In addition, this nitrosamine appears to be relatively more efficient at producing other kinds of mutations. In total 19 non-G:C----A:T mutations were identified. These included: A:T----G:C transitions, transversions and frameshifts. The relative contribution of these events was found also to decrease with increasing dose. These results may help explain why the parent carcinogen N-nitroso-N,N-dimethylamine is hepatotropic while other methylating carcinogens, similarly metabolized in the liver, are not.

Mutational specificities of environmental carcinogens in the lacl gene of Escherichia coli. II: A host-mediated approach to N-nitroso-N,N-dimethylamine and endogenous mutagenesis in vivo.

An intrasanguineous host-mediated assay was used to determine the mutational specificity of the hepatocarinogen N-nitroso-N,N-dimethylamine metabolized in vivo. A total of 114 forward mutations in the lacl gene of Escherichia coli reisolated from the livers of treated Swiss albino mice were characterized at the DNA sequence level. Consistent with the methylating ability of this compound and the demonstrated mutagenic specificity of O6-methylguanine, the predominant mutation was the G:C----A:T transition. These were recovered, on average, seven times more frequently at guanines flanked (5') by a purine residue than at those preceded by a pyrimidine residue--a specificity similar to that reported for many direct-acting SN1 alkylating agents. This nitrosamine appears to be distinguished from related N-nitroso methylating compounds by the induction of additional mutational events. Here, the exceptions consisted of four A:T----G:C transitions, four A:T site transversions, and a single G:C----T:A transversion. In addition, the DNA sequence alterations of 34 I- mutants of E. coli reisolated from otherwise untreated mice were identified. The predominant mutation was the G:C----A:T transition, which accounted for almost half of all background mutations. The sites at which these mutations were recovered appear to indicate that some of these mutations may have arisen as a result of an accelerated rate of cytosine deamination. These data suggest that many of the additional "spontaneous" mutations observed under in vivo conditions resulted from genotoxic events occurring during the host-defense (immune) reaction.

Mutation site specificity of N-nitroso-N-methyl-N-alpha-acetoxybenzylamine: a model derivative of an esophageal carcinogen.

A total of 171 mutations induced by N-nitroso-N-methyl-N-alpha-acetoxybenzylamine within the first 180 base pairs of the lacI gene of Escherichia coli were characterized at the DNA sequence level. Consistent with the methylating ability of this compound and the predicted mutagenic specificity of the O6-methylguanine lesion, all but two of these mutations were G:C----A:T transitions. An analysis of neighboring sequences revealed the same 5' flanking sequence influence on mutability reported for other SN1-type direct-acting alkylating agents. G:C----A:T transitions were found to be six times more likely to occur at G:C base pairs at which the guanine residues were flanked (5') by a purine than at those preceded by a pyrimidine. This mutagenic and site specificity appeared to be independent of the dose and likely reflects the behaviour of the activated parent carcinogen, N-nitroso-N-methyl-N-benzylamine in the esophagus.

Mutational specificity of thymine deprivation-induced mutation in the lacI gene of Escherichia coli.

LacI- mutants obtained following 2 and 6 h of thymine deprivation were cloned and sequenced. The mutational spectra recovered were dissimilar. After 2 h of starvation the majority of mutations were base substitutions, largely G:C----C:G transversions. Frameshift mutations but not deletions were observed. In contrast, following 6 h of starvation, with the exception of the G:C----C:G transversion, all possible base substitutions were recovered. Moreover, several deletions but no frameshift events were observed. The differences in the mutational spectra recovered after the two periods of thymine deprivation highlight the role of altered nucleotide pools and the potential influence of DNA replication and repair mechanisms.

Missense mutation in the lacI gene of Escherichia coli. Inferences on the structure of the repressor protein.

The lac repressor has been studied extensively but a precise three-dimensional structure remains unknown. Studies using mutational data can complement other information and provide insight into protein structure. We have been using the lacI gene-repressor protein system to study the mutational specificity of spontaneous and induced mutation. The sequencing of over 6000 lacI- mutations has revealed 193 missense mutations generating 189 amino acid replacements at 102 different sites within the lac repressor. Replacement sites are not distributed evenly throughout the protein, but are clustered in defined regions. Almost 40% of all sites and over one-half of all substitutions found occur within the amino-terminal 59 amino acid residues, which constitute the DNA-binding domain. The core domain (residues 60 to 360) is less sensitive to amino acid replacement. Here, substitution is found in regions involved in subunit aggregation and at sites surrounding residues that are implicated in sugar-binding. The distribution and nature of missense mutational sites directs attention to particular amino acid residues and residue stretches.

Nearest neighbor affects G:C to A:T transitions induced by alkylating agents.

The influence of local DNA sequence on the distribution of G:C to A:T transitions induced in the lacI gene of E. coli by a series of alkylating agents has been analyzed. In the case of nitrosoguanidine, two nitrosoureas and a nitrosamine, a strong preference for mutation at sites proceeded 5' by a purine base was noted. This preference was observed with both methyl and ethyl donors where the predicted common ultimate alkylating species is the alkyl diazonium ion. In contrast, this preference was not seen following treatment with ethylmethanesulfonate. The observed preference for 5'PuG-3' site over 5'-PyG-3' sites corresponds well with alterations observed in the Ha-ras oncogene recovered after treatment with NMU. This indicates that the mutations recovered in the oncogenes are likely the direct consequence of the alkylation treatment and that the local sequence effects seen in E. coli also appear to occur in mammalian cells.

Defect in excision repair alters the mutational specificity of PUVA treatment in the lacI gene of Escherichia coli.

The sequences of 152 lacI- mutations obtained following exposure of Escherichia coli UvrB- strain NR3951 to ultraviolet light in the presence of 8-methoxypsoralen (PUVA treatment) were compared to the spectrum of mutation induced by PUVA treatment in a Uvr+ strain, NR3835. Mutations recovered following PUVA treatment of the UvrB- strain were quite different from those recovered in the Uvr+ strain. In addition, they occurred at a restricted number of unique sites. For example, A.T----T.A base substitutions at position 141, minus G frameshifts at positions 586/587/588 and deletions of 15 base-pairs from position 78 to 92 accounted for 50% or more of mutations recovered in each of the above mutational classes. This altered mutational specificity was accompanied by a failure to recover mutations frequently identified following PUVA treatment of the Uvr+ strain. These mutations include spontaneous-hotspot frameshifts involving the gain or loss of a tetramer 5'-CTGG-3' repeated three times at position 620 to 631; and minus A.T base-pair frameshifts recovered at potential T-T crosslink sites. These results indicate that while crosslinks may play a substantial role in the induction of mutation in the Uvr+ strain, they do not contribute substantially to mutagenesis in the UvrB- strain. In addition, the data also suggest that excision repair may not always occur in an error-free manner.