Clonal analysis of childhood acute lymphoblastic leukemia with "cytogenetically independent" cell populations.

Acute lymphoblastic leukemia (ALL) is generally regarded as a clonal disease in which a single abnormal progenitor cell gives rise to neoplastic progeny. Five of 463 cases of childhood ALL with adequately banded leukemic cells were found to have two cytogenetically independent cell populations. In addition, two of the four cases tested had more than two rearranged immunoglobulin genes and (or) T cell receptor genes. To investigate the clonality of these unusual leukemias, we examined the neoplastic cells for X-linked markers extrinsic to the disease. Leukemic cells from each of the three patients heterozygous for an X-linked, restriction fragment length polymorphism showed a single active parental allele, suggesting that both apparently independent cell populations developed from a common progenitor. These cases provide evidence that leukemogenesis involves a multistep process of mutation and suggest that karyotypic abnormalities may be a late event of malignant transformation.

Wild-type p53 induction mediated by replication-deficient adenoviral vectors.

Replication-deficient E1-/E3-deleted adenoviral vectors are commonly used to introduce transgenes into cells in vitro and have been used for certain kinds of gene therapy protocols in vivo. We have demonstrated that transduction of cells using these vectors can induce p53 expression in cells containing a wild-type p53 gene. This response is different from p53 induction observed after DNA damage because the time course of induction is slower and because it occurs in cells with an attenuated DNA damage response. However, this vector-induced p53 is transcriptionally active and, therefore, p53 function is not inactivated by viral proteins. The mechanism of induction appears to be an increased rate of protein translation because immunoprecipitation analyses demonstrated increased levels of 35S-labeled p53 protein, even after a short 15-min labeling time. Induction of p53 by adenoviral vectors may have various effects on transduced cells, including apoptosis and altered chemotherapy chemosensitivity. Therefore, the influence of the vector might confound the impact of any particular gene used in a gene therapy application.

General and specific effects of amino acid starvation on the formation of undermodified Escherichia coli phenylalanine tRNA.

The heterogeneity of undermodified phenylalanine tRNA produced in relaxed control E. coli during amino acid starvation was investigated. Examination of the RPC-5 elution profiles of tRNAPhe prepared from non-starved cells and cells starved of a variety of amino acids, including some known to be involved in the formation of modified bases revealed that: (1) only one species of fully modified tRNAPhe appears to occur in cells grown in enriched medium; (2) at least two chromatographically unique isoacceptor species are observed in addition to the normal tRNAPhe in starved cells; (3) the unique, undermodified species of tRNAPhe from leucine-starved cells, known to be deficient in dihydrouridine, pseudouridine, 2-thiomethyl-N6-(delta2-isopentenyl) adenosine and 3-(3-amino-3-carboxypropyl) uridine, co-elute with the unique species produced in cells starved of histidine or arginine or treated with puromycin or chloramphenicol; (4) additional unique species of tRNAPhe can be detected in methyl- and sulfur-deficient tRNA from methionine- and cysteine-starved cells; (5) analysis of phenoxyacetylated tRNA revealed that the chromatographically unique and normal species from starved cells contain subspecies deficient in 3-(3-amino-3-carboxypropyl) uridine; and (6) using phenoxyacetylation as a means of effecting the resolution of undermodified subspecies, a total of at least ten chromatographically unique subspecies of rRNAPhe were detected in an organism that appears to posses only one gene for tRNAPhe. Taken together, the results support the view that there are both general and specific effects of amino acid starvation on the post-transcriptional modification of tRNA.

Residual disease detection in multiple follow-up samples in children with acute lymphoblastic leukemia.

The polymerase chain reaction (PCR) is more sensitive than other methods for detecting residual leukemic cells, and it can be used to analyze the dynamics of the leukemic cell population during and after chemotherapy. Leukemia cells are identified among the normal cells through the use of a leukemic cell-specific signature, such as the junction formed following rearrangement of the immunoglobulin (Ig) or T-cell receptor (TCR) variable (V), diversity (D), and joining (J) regions. We cloned the rearranged Ig or TCR genes from leukemic cells at the time of diagnosis by regular recombinant DNA techniques, or following PCR amplification, and determined the leukemic cell-specific V(D)J junctions. Using oligonucleotides prepared to the unique junctional sequences, we have analyzed Southern blots of PCR-amplified Ig or TCR genes from 11 patients at the time of diagnosis, and in from three to 14 follow-up samples for periods ranging from 17 to 97 months. The level of detection of residual blast cells in these patients was usually on the order of 1 leukemic cell in 10(4) normal ones. Conditions for the hybridization and washing were individually adjusted so that the specificity of the probes was excellent. No residual leukemic cells were found in patients who remained in continuous complete remission at time points more than 6 months post-diagnosis. PCR detected impending relapse in four out of six cases, with PCR-detectable leukemic clones being present 2, 4, 13 and 20 months prior to clinical signs of the disease. Relapse occurred in two patients who lacked PCR evidence of leukemic blasts in marrow samples collected 2 and 6 months before clinical recurrence, respectively. The leukemic cells that persisted for 3 years after a relapse in a patient who remained in clinical remission during this time period eventually became undetectable by PCR.

Characterization of immunoglobulin heavy chain genes from an acute lymphocytic leukemia with four rearrangements.

Approximately 25% of acute leukemias of the B-cell lineage demonstrate more than two rearranged immunoglobulin heavy chain genes when examined by Southern blot analysis. The origin of the extra bands was investigated by molecular cloning and sequencing of four rearranged genes from one patient's leukemic cells. All four rearrangements were apparently derived independently. Two of the rearrangements used the VH6 variable region, attached to different diversity and joining regions. One of the two rearrangements contained a mutation in the coding sequence leading to the generation of a nonsense codon. This rearranged gene also differed from the other VH6 containing gene starting at about 330 bp upstream of the ATG initiation codon. The third rearranged gene used a member of the VH2 variable gene family. A DH-JH rearrangement was found in the fourth rearranged gene. The data indicate that the leukemia probably arose as a result of the transformation of an early B-cell progenitor that lacked rearranged immunoglobulin genes but retained some differentiation potential.

Alterations in immunoglobulin or T cell receptor gene rearrangement at relapse: involvement of 11q23 and changes in immunophenotype.

We studied the organization of immunoglobulin (Ig) genes and the beta-chain gene of the T cell receptor (TCR) along with the clinical features, immunophenotypes, and karyotypes of 14 children with acute leukemia at diagnosis and relapse. The median time to relapse was 23 months. Eleven children had identical gene rearrangement patterns at diagnosis and relapse. All three patients whose blast cells showed variations in gene rearrangement patterns between diagnosis and relapase also demonstrated a change in the immunophenotype: one from cALLA+ to cALLA- B precursor cell ALL; one from T-ALL to AML; and one showed a marked increase in myeloid characteristics at relapse. Blast cells from these three patients at relapse showed the presence of chromosomal translocations involving 11q23; for two patients, this involved replacement of the original karyotype. We conclude that while most relapses are the result of reemergence of the original clone, new clones that differ in immunophenotype, karyotype, and gene rearrangement are occasionally present at relapse. Relapses may also occur in which the original clone has undergone major changes in gene expression and arrangement of the Ig heavy chain genes in the absence of karyotypic replacement.

Minimal residual disease after intensive induction therapy in childhood acute lymphoblastic leukemia predicts outcome.

We investigated the level of minimal residual disease (MRD) in 26 children with B-lineage acute lymphoblastic leukemia (ALL) after intensive induction therapy. A quantitative semi-nested polymerase chain reaction (PCR) detecting the clone-specific rearranged immunoglobulin heavy chain genes was developed to improve sensitivity and specificity of amplification. In all patients, one leukemic cell could be detected in a background of 10(5) normal blood mononuclear cells. All patients investigated were in complete remission at the end of induction therapy as evaluated by morphologic criteria. Nineteen patients (73%) had no detectable residual leukemic cells using the sensitive semi-nested PCR. Seven patients (27%) were PCR positive. Three had a low level (<2 x 10(-5) leukemic cells per bone marrow cell), while four patients had a high level (>2 x 10(5)) of detectable residual leukemic cells. All patients with low or undetectable levels of residual leukemia remained in complete remission at a median of 63 months from diagnosis (range 40-80 months), while all four patients with a high level of residual leukemia subsequently relapsed at a median of 21 months from diagnosis (range 13-37 months). The patient groups with undetectable or low, and high level of MRD did not differ significantly in other clinical or genetic features with prognostic significance. We conclude that the level of MRD at the end of the intensive induction therapy period is predictive of outcome in childhood B lineage ALL. If confirmed by large prospective studies, the level of MRD might be useful in stratifying patients into high and low risk categories.

Extensive cross-reactivity of adenovirus-specific cytotoxic T cells.

Although adenovirus is a major source of morbidity for immunocompromised individuals and a popular vector for gene therapy, little is known about the cellular immune responses it evokes in humans. Initial trials using adenovirus vectors have been disappointing, probably owing both to a preexisting immune response to Ad2 and Ad5, the most commonly used vector backbones, and to a response to the transgene. The former problem might be overcome by switching from the common type C adenoviruses, of which Ad2 and Ad5 are members, to other less common serotypes. Evidence for the feasibility of this approach has been provided by a rat model system. However, its success in humans depends on there being no immunological cross-reactivity between groups at the humoral or cellular level. Here, we examine the cross-reactivity of the cellular immune response to adenovirus in a human system, and find that human cytotoxic T lymphocytes (CTLs) prepared in vitro against an adenovirus from two of the six subgroups can lyse cells infected with adenoviruses from the other subgroups. Hence, the proposed use of adenovirus vectors from uncommon subgroups to evade memory immune response to subgroup C adenoviruses may not be successful. However, this same cross-reactivity indicates that adoptive transfer of CTLs generated in vitro against one adenovirus serotype may protect immunocompromised patients from infections by adenoviruses of all serotypes.

Restriction mapping and molecular cloning of adenovirus type 4 (subgroup E) DNA.

The locations of thirty restriction endonuclease cleavage sites were determined on the genome of adenovirus type 4 (Ad4), the sole member of the subgroup E adenovirions. The restriction endonucleases Bg/II, EcoRI, HindIII, HpaI, KpnI, SalI, and XbaI cut Ad4 DNA 10, 3, 2, 3, 5, 5 and 3 times, respectively. Orientation of the linear Ad4 map with respect to left and right molecular ends was accomplished by taking advantage of the limited sequence homology between Ad2 and Ad4. Ten non-overlapping fragments of Ad4 DNA representing 98% of the genome, map units 1.6 to 99.6, have been cloned into the plasmid vector pKC7.

Isolation of activated ras transforming genes from two patients with Hodgkin's disease.

Despite impressive advances in the clinical management of Hodgkin's disease, little is known about its cellular origin or the mechanism(s) of "Hodgkinogenesis." Recent findings that certain human cellular oncogenes can cause malignant transformation suggest that aberrant activation of these genes may play a role in carcinogenesis. To determine if such genes are operative in Hodgkin's cells, we isolated DNA from splenic nodules of three patients with nodular sclerosis Hodgkin's disease and tested its ability to transform mouse NIH 3T3 cells, the standard assay for oncogene-mediated malignant transformation. Transformed cells containing human DNA were obtained from two patients. DNA from these primary transformants yielded secondary transformants of NIH 3T3 fibroblasts; one also transformed normal mouse bone marrow macrophages, a cell type probably related to the Hodgkin's cell. When analyzed by Southern blot methods for homology with closed oncogene probes, the transforming genes from both patients had homology with N-ras. The homology and size of the restriction fragments were similar to those of transforming genes isolated from patients with acute nonlymphocytic leukemias. The presence of the same activated oncogene in tumor tissue from two different patients suggests that it may play an important role in Hodgkinogenesis.

The clinical significance of residual disease in childhood acute lymphoblastic leukemia as detected by polymerase chain reaction amplification by antigen-receptor gene sequences.

The polymerase chain reaction (PCR) has been applied to detect occult leukemia cells in children with acute lymphoblastic leukemia who are otherwise considered in complete remission by traditional morphological examination of bone marrow specimens. To determine whether PCR provides unique prognostic information of use for the clinical investigator, we reviewed the 20 clinical studies published to date. From this review, it is evident that discrepancies exist for the detection of residual disease for patients who remain in complete remission and for those who relapse. However, because of the fundamentally different approaches used to apply the PCR method to each of these studies, an entirely different interpretation can be reached when critical technical factors are considered. The combined data from the various studies suggest that a consistent pattern for residual disease disappearance over many months exists for patients who remain in extended complete remission and a pattern of residual disease persistence and reappearance preceding clinical findings exists for the majority of those who ultimately relapse in the bone marrow.

Detection of minimal residual disease in all: biology, methods, and applications.

The PCR technique appears to be the most sensitive method for detecting residual disease in ALL and can be applied to a high percentage of cases by amplifying sequences of the antigen-receptor genes. The PCR studies to date suggest that this sensitive technique can detect residual disease in virtually all patients during the first year of treatment. The residual disease becomes undetectable in the majority of patients by the end of treatment; however, a subset of patients remain PCR positive at a time when therapy is electively discontinued. The development of a highly accurate quantitative PCR technique may allow the possibility of distinguishing the patterns of residual disease for patients who will be cured by treatment from those who relapse. If such a pattern can be discerned, then an immediate benefit for PCR monitoring will be that clinicians will have the opportunity to test whether treating patients at the time of 'molecular relapse' will help to improve the cure rate for this disease. The PCR studies of remission marrows at the end of treatment raise a number of questions about the biology of disease persistence in patients who remain in extended 'remission.' A commitment to obtaining and analyzing bone marrow specimens in patients who have completed therapy is necessary to discern whether novel strategies, such as immunomodulatory manipulations, are needed to control or eradicated residual disease in patients who have completed planned chemotherapy. Thus, the long-term benefit of residual disease monitoring by PCR may be a better understanding of the biology and definition of 'cure' in ALL.

Sequencing of selected regions of the human immunoglobulin heavy-chain gene locus that completes the sequence from JH through the delta constant region.

Much of the nucleotide sequence between the start of the joining region and the end of the immunoglobulin heavy chain delta gene has already been determined. However, two gaps existed in potentially functionally important regions in this sequence: the region between the 3' end of the joining region and the heavy chain enhancer region and that between the enhancer and the mu constant region. We have determined the nucleotide sequences of these regions. The 734 bp between the joining and enhancer regions contained no additional joining regions. The 4525 bp region between the heavy chain enhancer and the mu constant region contains the mu switch region, which consists of pentameric repeats. Approximately 60% of these repeats are GGGCT and GAGCT. With the determination of these sequences, the entire region of the heavy chain locus starting upstream of the joining region to downstream of the last exon of the delta constant region (a total of more than 29 kb) has now been sequenced.

Immunoglobulin heavy chain gene VH-D junctional diversity at diagnosis in patients with acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) represents the clonal outgrowth of transformed hematopoietic progenitor cells. We have found that blast cells in some cases of B-precursor cell ALL contain Ig heavy chain gene rearrangements with considerable diversity at the junctions of the variable (VH), diversity (D), and joining (JH) regions. This diversity consists of heterogeneous nucleotide sequences at the VH-D and, less frequently, the D-JH junctions. In two cases, different VH segments were attached to the same D-JH rearrangement. In all cases studied there was a much higher than expected frequency of nucleotide sequence changes in the VH segment. At least three mechanisms may produce these changes in different cases: (1) continuing rearrangement of the heavy chain gene, in some cases by VH addition to a preexisting D-JH; (2) VH replacement; and (3) an open-and-shut mechanism. These findings suggest that an active VDJ recombinase system is present at the time of transformation in a high percentage of ALLs. An active recombinase in the rapidly growing leukemic cell population could lead to genomic instability.

Mutations in the adenovirus-encoded single-stranded DNA binding protein that result in altered accumulation of early and late viral RNAs.

Adenovirus encodes a 72-kDa single-stranded DNA binding protein (DBP) that is necessary for viral DNA replication and is involved in controlling viral RNA metabolism. Studies of temperature-sensitive (ts) and site-directed mutants of the DBP have identified at least four regions of the protein involved in binding to single-stranded DNA and hence in DNA replication. Two of the ts mutants, Ad2ts111A and Ad2ND1+ts23, are deficient in DNA binding and in supporting in vitro DNA replication. Their effects on viral RNA metabolism are presented here. At early and late times of infection, accumulation of RNAs from the viral E3 region is increased up to sevenfold in Ad2ts111A- and Ad2ND1+ts23-infected cells relative to wild-type virus. This effect is temperature-independent and seems to involve nuclear RNA stability. Steady-state levels of the viral E1B and E4 RNAs increase following the onset of viral DNA replication in cells infected by the wild-type virus, but not in cells infected by ts111A or ts23. The increase in E1B and E4 RNAs at late times of infection is due to a stabilization of the mRNA. The steady-state levels of L3 and L5 RNAs are two- to fourfold higher in cells infected with ts23 and ts111A than with wild-type virus. None of these differences were observed following infection of cells with a temperature-independent revertant of ts111, indicating that the mutations in the DBP were responsible for the phenotypes. However, for the E3 effect, the change brought about by the mutations in the DBP does not seem to be the result of a change in the normal function of the protein, as an essentially DBP-negative virus (Ad5d/802) shows no differences in E3 RNA metabolism compared to wild-type virus at early times of infection. These results demonstrate that mutation of amino acids 280 and 282 of the DBP significantly perturbs the normal regulation of viral RNA metabolism. These effects clearly differ from the phenotypes observed with adenoviruses containing mutations in other early genes and from those ascribed to the ts125 mutation by others. These results are discussed in terms of the mechanisms by which early and late viral RNA metabolism are controlled and the possible effects of the DBP mutations on them.

Sequence of the DNA-binding protein of a human subgroup E adenovirus (type 4): comparisons with subgroup A (type 12), subgroup B (type 7), and subgroup C (type 5).

The nucleotide sequence of the gene for the single-stranded DNA-binding protein of adenovirus type 4 (Ad4) has been determined. The gene codes for a protein of 512 amino acids. Comparison of the amino acid sequence with those previously determined for Ad5, Ad12, and Ad7 allowed identification of regions that are conserved between the four serotypes. These include stretches of 9, 9, and 12 amino acids in the carboxy-terminal domain of the protein; these sequences are similar to those identified in the single-stranded DNA-binding proteins of procaryotes as being important for interaction of the protein with single-stranded DNA. A conserved region of four amino acids in the amino-terminal domain is identical in sequence to a region of the SV40 large T antigen that has recently been implicated in the nuclear localization of the protein. Other conserved amino acids that may be important for the three-dimensional structure of the protein have also been identified. The overall homology between the DBPs of the four serotypes is 17.2% in the amino-terminal domain, 47.8% in the carboxy-terminal domain. Two-way comparisons between the DBPs of the four serotypes indicates that the DBP of Ad4 is most closely related to that of Ad7.

mRNA transcripts initiating within the human immunoglobulin mu heavy chain enhancer region contain a non-translatable exon and are extremely heterogeneous at the 5' end.

Transcription events are thought to precede gene rearrangement in the immunoglobulin (Ig) loci and may be the mechanism by which the various gene regions are made accessible for recombination. If this is the case, identification and characterization of transcripts from the Ig loci should permit a better understanding of the gene rearrangement process. We have isolated a 2.3 kb cDNA clone from the human pre-B cell line Nalm-1 that contains enhancer-specific sequences from the Ig heavy (H) chain gene locus. The 2.3 kb transcript initiated within the enhancer region and showed extreme 5' heterogeneity, with more than 50 initiation sites mapping near the Ig-specific octamer ATTTGCGT. Sequencing of the cDNA clone demonstrated that 644 nucleotides from the Ig enhancer region were incorporated as a leader exon spliced to the mu constant (Cmu) region. This leader exon contained many translation termination codons and may function to inhibit the translation of sterile Cmu polypeptides. Using an enhancer-derived probe, we detected two low-abundancy mRNA transcripts with sizes of 2.3 and 12 kb. Northern blot analysis suggested that the 12 kb transcript was the unspliced precursor mRNA of a VDJ rearrangement. The potential role of these enhancer-containing transcripts in the opening of the IgH chain gene for rearrangement and for class switching is discussed.

Unbalanced growth and the production of unique transfer ribonucleic acids in relaxed-control Escherichia coli.

The unique leucine-, arginine-, valine-, and phenylalanine-specific transfer ribonucleic acids (tRNA's) produced in relaxed-control (rel-) Escherichia coli during leucine or arginine starvation are chromatographically similar to those produced by chloramphenicol treatment. The major unique rel- leucine-specific and phenylalanine-specific tRNA's are heterogeneous, accumulate with time of starvation, and can account for up to 70% of the respective amino acid acceptor activities. The changes which occur in the isoacceptor profiles for tRNALeu and tRNAPhe as a function of starvation time suggest that the unique species are undermodified precursors to the major isoacceptor species observed in nonstarved cells. Analyses of the isoacceptor patterns of tRNA from cells recovering from starvation suggest that the unique species of tRNALeu and tRNAPhe may not be normally occurring precursors. When leucine-starved cells were incubated in fresh, fully supplemented medium, the major unique tRNALeu and tRNAPhe appeared to be converted to normal species only slowly or not at all. The results are consistent with the view that some of the events in the post-transcriptional modification of tRNA may occur in an ordered sequence. An examination of the subcellular distribution of the unique leucine and phenylalanine tRNA's revealed that these species occur on the ribosome at about the same frequency as the major, normally occurring isoacceptor species. This result provides additional evidence of a precursor-product relationship for the unique and normal tRNA's and further indicates that there is no discrimination against the unique species by the ribosome.

Analysis of the imperfect octamer-containing human immunoglobulin VH6 gene promoter.

The octamer sequence ATGCAAAT is highly conserved in the promoter of immunoglobulin heavy and light chain genes and is one of the sequence motifs involved in the control of transcription of these genes. The promoter region of an human immunoglobulin heavy chain variable gene, the sole member of the VH6 gene family, was found to differ from other VH gene promoters: it contains neither the conserved octamer motif nor a heptamer sequence, and generally bears little resemblance to other VH gene transcriptional control regions. An imperfect octamer sequence with a single nucleotide substitution (AgGCAAAT) is located 108 bp upstream of the ATG translation start site, and 81 bp upstream of the transcription initiation site. We sought to determine which sequence elements within the VH6 promoter were responsible for transcription initiation by creating progressive deletions of a 1 kb fragment from this region and testing their ability to function as promoter elements in B and non-B cells (HeLa). The minimum fragment required for full promoter function was 110 bp, but a fragment with only 65 bp retained 30-50% activity in B cells. Similar levels of transcription were seen when the -146 bp promoter containing two point mutations in the imperfect octamer was tested. Mutation of a possible pyrimidine box sequence located downstream of the TATA box was shown to have only a minor effect (10-30%) on transcription when three nucleotides were changed. Surprisingly, CAT activity was not B cell-specific, as all constructs had virtually the same activity in several B cell lines and in HeLa cells. Removal of the TATA box led to a 50% reduction in CAT activity, and the region upstream of the TATA box functioned as a promoter in both orientations. The transcriptional activity of the VH6 promoter was virtually enhancer independent: only a minor increase was observed when the immunoglobulin or SV40 enhancer was added to the promoter construct. Electrophoretic mobility shift assays of transcription factor binding to the region around the imperfect octamer indicated that binding was weak when nuclear extracts from either B cells or HeLa cells were used. The amount of complex shifted was increased by mutating the imperfect octamer to a perfect one. Chimeras produced between the VH6 promoter and a B cell-specific promoter from a member of the human VH2 gene family demonstrated that the lack of tissue specificity was due to the absence of a repressor of non-B cell transcription in the VH6 promoter. These results indicate that the VH6 promoter is relatively simple, requiring little more than the TATA element and the imperfect octamer, and transcription from this promoter lacks B cell specificity and is not dependent on the enhancer element.