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Biomedical subjects

G Rovera

Publications and source records attributed to G Rovera.

At least 19 recordsLinked to original sources
PubMed1992

Predominance of fetal type DJH joining in young children with B precursor lymphoblastic leukemia as evidence for an in utero transforming event.

The presence of N sequences in the complementarity determining region 3 (CDR3) of the rearranged immunoglobulin H chain is developmentally regulated: N regions are generally present in the DJH joinings of adult B cells but are often absent in fetal B cells. Analysis of the CDR3 in 61 B precursor acute lymphoblastic leukemias indicated that 87.5% of the leukemias obtained from children < or = 3 yr old lacked N regions at the DJH junction. In contrast, in children > 3 yr old, only 11.1% of the leukemias lacked N regions at this junction, a frequency similar to what we have observed in B cells from children and adults. These findings suggest that the majority of leukemias presenting within the first 3 yr of age arise from an in utero transforming event.

Adult↗
PubMed1992

Selective inhibition of proliferation in v-abl- and bcr-abl-transformed cells by a nucleoside analog.

The nucleoside analog acyclovir (9-[2-hydroxy-ethoxy)methyl]guanine or acycloguanosine; ACV) inhibited the in vitro transformation of NIH 3T3 cells by Abelson murine leukemia virus and the proliferation of abl- and bcr-abl-transformed hemopoietic murine cell lines. This effect is selective since ACV at the same concentration had no effect on the src and Ha-ras transformation of NIH 3T3 cells or on the proliferation of hemopoietic cells transformed by those oncogenes. The inhibitory effect on proliferation of abl-transformed cells correlated with the extent of ACV triphosphate formation and incorporation into cellular DNA that was greater than that in normal or other oncogene-transformed cells. The increased ACV triphosphate formation might be due to a higher level of 5'-nucleotidase, the enzyme responsible for trace levels of ACV phosphorylation in uninfected cells.

3T3 Cells↗
PubMed1992

The pattern of joining (JH) gene usage in the human IgH chain is established predominantly at the B precursor cell stage.

Preferential utilization of JH and D genes has been demonstrated in the rearranged IgH chain in human peripheral B cells. We report here that the same hierarchy of JH gene usage is observed in leukemic cells arrested in the B precursor stage of differentiation. Specifically, JH4 and JH6 accounted for 42.9% and 35.7%, respectively, of the JH gene usage in the leukemias compared with an expected frequency of 16.7% assuming unbiased gene usage. Within the D gene families, the DN1 gene appears to be overutilized in both populations, representing about 15% of the total gene usage compared with an expected frequency of 3.2%. Because 21 of the 36 leukemias contained only nonproductive IgH rearrangements, the preferential gene usage could not have arisen from pre-B cells that have undergone clonal selection after a productive rearrangement but before surface Ig expression. Nonproductive rearrangements exhibited the biased gene usage seen for productive rearrangements. These findings suggest that a recombination bias favoring certain segments may be the actual mechanism responsible for the apparent preferential utilization of JH and D genes.

Adolescent↗
PubMed1992

Inhibition of myeloid differentiation by the helix-loop-helix protein Id.

Id is a helix-loop-helix (HLH) protein that represses activity of several basic helix-loop-helix (bHLH) proteins involved in cell type--specific transcription and cell lineage commitment. The myeloid precursor cell line 32DC13(G) expressed Id messenger RNA, which was transiently decreased when cells were induced to terminally differentiate with granulocyte--colony-stimulating factor. Concomitant with the decrease of Id messenger RNA was the appearance in nuclear extracts of DNA binding proteins that recognized a canonical E-box motif, a DNA binding site for some bHLH proteins. Constitutive expression of an Id complementary DNA in 32DC13(G) cells blocked their ability to differentiate and to induce E-box-binding activity. These results suggest that Id and, hence, bHLH proteins function in the process of myeloid differentiation.

Cell Differentiation↗
PubMed1992

Structure of the murine lactotransferrin gene is similar to the structure of other transferrin-encoding genes and shares a putative regulatory region with the murine myeloperoxidase gene.

The structure and nucleotide sequence of the murine lactotransferrin-encoding gene (LTF) deduced partly by direct sequencing of genomic clones in the lambda phage vector and partly by enzymatic amplification of genomic DNA segments primed with the oligodeoxyribonucleotide primers homologous to the cDNA sequence. The lambda phage clones contained the 5' half of the gene corresponding to the first eight exons and an incomplete ninth exon interrupted by eight introns. Genomic clones corresponding to the 3' half of the LTF gene could not be obtained on repeated attempts from two different mouse genomic libraries, suggesting the possible presence of unclonable sequences in this part of the gene. Hence, PCR was used to clone the rest of the gene. Four out of the presumed eight remaining introns were cloned along with the flanking exons using PCR. Comparison of the structure of the LTF gene with those of the two other known transferrin-encoding genes, human serum transferrin-encoding gene and chicken ovotransferrin-encoding gene reveals that all three genes have a very similar intron-exon distribution pattern. The hypothesis that the present-day transferrin-encoding genes have originated from duplication of a common ancestral gene is confirmed here at the gene level. An interesting finding is the identification of a region of shared nucleotides between the 5' flanking regions of the murine LTF and myeloperoxidase-encoding genes, the two genes expressed specifically in neutrophilic granulocytes.

Animals↗
PubMed1992

VH gene rearrangement events can modify the immunoglobulin heavy chain during progression of B-lineage acute lymphoblastic leukemia.

The presence of multiple VHDJH joinings in upwards of 30% of acute lymphoblastic leukemias (ALL) suggests a relative instability of the rearranged immunoglobulin heavy chain (IgH) gene, but the mechanisms involved are not completely understood. An investigation of the structure of the VHDJH joinings using complementarity determining region (CDR)3 polymerase chain reaction (PCR) in 12 leukemias at both diagnosis and relapse indicates that this instability may increase as a function of time. In only one of seven cases in which relapse occurred within 3 years from diagnosis was a new VHDJH joining identified and this coexisted with the original diagnostic joining. Most strikingly, new VHDJH joinings were identified in four of five cases in which relapse occurred more than 5 years from diagnosis. In this latter population, the instability of the joinings was generated from VH----VH gene replacement events in two cases, since the new joinings retained the original DJH sequences and partial N region homology at the VHD junction, and probably in a third case from a VH gene rearrangement to a common DJH precursor. Furthermore, in five of 23 (21.7%) additional cases studied at diagnosis, subclones were identified that had similar modifications of the VH-N region. These data indicate that VH gene replacement events and VH gene rearrangements to a common DJH joining contribute to the instability of the VHDJH joining in ALL. This phenomenon should be taken into consideration in those methodologies that exploit IgH rearrangements for detection of minimal residual disease.

Adolescent↗
PubMed1992

Residual disease at the end of induction therapy as a predictor of relapse during therapy in childhood B-lineage acute lymphoblastic leukemia.

PURPOSE: More than 95% of children with B-lineage acute lymphoblastic leukemia (ALL) achieve a clinical remission after the induction phase of chemotherapy (first 28 days) as evaluated by morphologic criteria. However, relapse occurs in approximately 30% of these children. The objective of this study was to determine whether the outcome of patients in clinical remission at the end of induction therapy could be predicted using a highly sensitive method to detect residual disease. PATIENTS AND METHODS: All children diagnosed with B-lineage ALL at the Children's Hospital of Philadelphia during a 2-year period were eligible. The extent of residual leukemia was quantitated in remission marrow samples obtained at the end of induction therapy in 44 children using a phage clonogenic assay in association with complementarity-determining-region 3 (CDR3)-polymerase chain reaction (PCR). RESULTS: Residual disease was a significant predictor of outcome independent of WBC count, age, or sex. The estimated relapse-free survival (RFS) during therapy was 50.4% (+/- 12.6%) for patients with high residual disease (> or = 0.6% leukemia cells among total marrow B cells) versus 91.9% (+/- 5.5%) for those with lower levels (P < .002). There were no significant differences in off-treatment RFS between patients with high or low residual disease who completed therapy in continuous remission (P = .82). The overall estimated RFS was 32.3% (+/- 11.6%) for patients with high residual disease versus 62.6% (+/- 10.7%) for patients with lower levels of residual leukemia cells, with a median follow-up of 5.3 years for patients in continuous remission (P < .008). CONCLUSION: PCR detection of high residual disease at the end of induction therapy identifies patients at increased risk for relapse during therapy.

Adolescent↗
PubMed1992

The immediate early gene response to a differentiative stimulus is disrupted by the v-abl and v-ras oncogenes.

The immediate early gene activation in response to a differentiative stimulus was investigated in the hematopoietic progenitor cell line 32DC13(G). A transient and coordinated increase in mRNA levels for c-fos, c-jun, jun-B, TIS-7/PC-4, TIS-8/egr-1, and TIS-11 occurred during the first 2 h of treatment with granulocyte colony stimulating factor (G-CSF), which ultimately induces the 32DC13(G) cells to terminally differentiate into neutrophilic granulocytes. This pattern of mRNA induction was disrupted by v-abl and v-ras, two oncogenes known to interfere with G-CSF-induced differentiation of 32DC13(G) cells. Induction of the mRNAs for c-jun and TIS-7/PC-4 was blocked by the presence of v-abl, whereas v-ras caused constitutive expression of c-fos mRNA and blocked the c-jun, jun-B and TIS-7/PC-4 mRNA response. Release of the differentiation block in the ras-transformed 32DC13(G) cells by co-treatment with retinoic acid and G-CSF partially restored the normal c-fos and c-jun mRNA induction pattern, suggesting that the proper activation of these genes may be important for myeloid differentiation.

Cell Differentiation↗
PubMed1992

The family in different sub-cultures.

The family has been considered, according to the different angles from which research has been conducted, as an anthropological reality; as an institutional segment; as an etiopathogenetic nucleus; as a communicative environment. The family, taken as a highly complex (open-closed) system, is by now a common conception not only among family therapists, but also for those concerned--from a transcultural point of view--with individual and family dynamics in connection with extra-familial dynamics in a society in constant and rapid transformation. The therapists must be able to contribute to a reconstruction of the interactive dynamics of the family network, stimulating the contribution that each member may make to reparative, and at the same time creative, development. In the various family typologies the model of a therapeutic network flexibly based on cultural identification may be introduced.

Culture↗
PubMed1991

Transcriptional regulation of two myeloid-specific genes, myeloperoxidase and lactoferrin, during differentiation of the murine cell line 32D C13.

The transcriptional regulation of myeloperoxidase (MPO) and lactoferrin (LF) was examined during terminal myeloid differentiation of the murine cell line 32D C13. The rates of transcription initiation for MPO and LF, determined by an in vitro nuclear run-on assay, increased approximately ninefold. The accumulation of MPO mRNA in 32D C13 cells, determined by Northern blot analysis, correlated temporally with the observed increase in MPO transcription initiation. On the other hand, accumulation of LF mRNA lagged behind the observed increase in LF transcription initiation. In mouse L cells, the LF gene was transcribed more frequently than the MPO gene, though neither mRNA accumulated. Finally, murine MPO transcription is shown, by Northern blot and primer extension analysis, to initiate at multiple sites. These results indicate that whereas transcription induction may largely account for the accumulation of MPO mRNA during terminal myeloid differentiation, both transcriptional and posttranscriptional mechanisms operate to allow accumulation of LF mRNA. The 32D C13 cell system will be a useful model for elucidating these mechanisms.

Animals↗
PubMed1991

Homing and progression patterns of childhood acute lymphoblastic leukemias in severe combined immunodeficiency mice.

The aim of this study was to analyze the homing and progression patterns of childhood acute lymphoblastic leukemias (ALL) in mice with severe combined immunodeficiency (SCID). Upon intraperitoneal (IP) transfer, cells from relapse samples of three children with T-lineage ALL spread hematogenously and infiltrated the non-lymphoid and/or lymphoid organs with a pattern reminiscent of the human clinical disease. These mice either died or were killed in extremis at a mean of 9 weeks. Moreover, cell lines established in vitro from two of these samples manifested identical homing and progression in the SCID mouse as compared with the original patients' cells. Thus, long-term culture of the primary leukemic T cells did not alter their invasive potential and migration pattern. When engrafted IP, three cell lines established from pre-B-ALL cases displayed primarily a lymphatic spread with induction of local tumor masses and kidney/liver nodules. Mice were killed at 11 to 13 weeks, but had not developed imminently fatal leukemia. However, when transferred intravenously, one pre-B ALL cell line was able to spread hematogenously and to infiltrate both lymphoid and non-lymphoid tissues. Overall, these data demonstrate that the SCID mouse provides an efficient and reproducible model to study the pathogenesis of childhood ALL, and may be a suitable system for evaluating therapy.

Animals↗
PubMed1991

Growth factor requirements of childhood acute T-lymphoblastic leukemia: correlation between presence of chromosomal abnormalities and ability to grow permanently in vitro.

Cells from 10 cases of childhood acute T-lymphoblastic leukemia (T-ALL) were cultured in the presence of recombinant human interleukins (rhIL) or colony-stimulating factors (CSF) to analyze their growth factor requirements and differentiative potential. Although cells from most leukemic samples displayed a short-term proliferative response to several hematopoietic growth factors, only the ones featuring chromosomal translocations could be established as permanent cell lines. Two cell lines could be initiated only in the presence of IL-3 (TALL-103 and TALL-106), one in granulocyte-macrophage CSF (GM-CSF) (TALL-101), and one in IL-2 (TALL-104); only one cell line (TALL-105) was originated in the absence of growth factors. The TALL-101 and TALL-103 cell lines, derived from very immature T-ALL cases, underwent growth factor-dependent phenotypic conversion (lymphoid to myeloid). However, the T-cell receptor rearrangement and karyotype of the original leukemic clones were retained. In contrast, the TALL-104, -105, and -106 cell lines which originated from more mature T-ALL cases, maintained a T-lymphoblastic phenotype regardless of the growth factors in which they were expanded. These data demonstrate in vitro the aggressive nature of T-ALL cases bearing chromosomal abnormalities, and indicate that the lineage commitment of the malignant clone depends on its stage of maturation in T-cell ontogeny.

Adolescent↗
PubMed1991

Preferential utilization of specific immunoglobulin heavy chain diversity and joining segments in adult human peripheral blood B lymphocytes.

We have examined at the molecular level the CDR3 and adjacent regions in peripheral blood B lymphocytes of normal individuals. A total of 111 sequences (12-28 sequences from six individuals) were obtained after cloning of the polymerase chain reaction-amplified segments into plasmids or phage. The average length of the VDJ joining was 109 nucleotides, with a range from 79 to 151. Approximately 75% of the sequences were in frame when translated into amino acids. Among the JH segments, JH4 was found most frequently (in 52.5% of the sequences), and JH1 and JH2 segments the least frequently (approximately 1% of the clones). A polymorphic JH6 gene with a one-codon deletion accompanied by a base change was present in two of six patients. Preferential breakpoints were found for JH2, JH3, JH4, and JH5, although the breakpoints of JH6 were distributed more heterogenously. In approximately 90% of the cases, significant homology of the D regions with published D sequences was found. Preferential usage of a particular coding frame was observed in in-frame sequences utilizing DA, D21/9, and DM1 segments. However, in general, all coding frames of germline D genes were used to generate CDR3s. Eight sequences that have a DN1-like D sequence with two base changes at the same positions were identified, suggesting the likely existence of a new germ line D gene belonging to the DN families. Using probes specific for a particular CDR3, the frequency of a specific B cell clone in the peripheral blood of normal individuals was estimated to be at most as high as 1/20,000.

Amino Acid Sequence↗
PubMed1991

Molecular diagnosis of cutaneous T-cell lymphoma: polymerase chain reaction amplification of T-cell antigen receptor beta-chain gene rearrangements.

The goal of our study was to molecularly diagnose CTCL, by cloning the T-cell antigen receptor beta chain (TCR-beta) gene rearrangement from the malignant T cells of a patient with Sézary syndrome, in order to generate a specific oligonucleotide probe capable of detecting CTCL cells through polymerase chain reaction (PCR) amplification. Total RNA isolated from peripheral blood lymphocytes was reverse transcribed and resultant first strand cDNA was PCR amplified utilizing a concensus primer to the TCR-beta variable region (V beta) and a 3' primer to the TCR-beta constant region (C beta). PCR reaction products were subcloned into a plasmid vector and sequenced. Sequence analysis revealed that the patient's in-frame TCR-beta gene rearrangement utilized V beta 6.4, D beta 1.1, J beta 2.2, and C beta 2.1 gene segments. Oligo-primers to V beta 6.4 and J beta 2.2 were utilized to PCR amplify genomic DNA taken from the patient's blood and involved skin. Screening the amplified DNA with an oligo-probe specific for the patient's V-D-J junctional sequences resulted in the detection of the patient-specific sequences. No sequences were detected from DNA from other malignant or benign infiltrates. Thus, we have defined a "molecular fingerprint" specific for a patient's malignant T-cells and can molecularly diagnose CTCL through PCR amplification.

Amino Acid Sequence↗
PubMed1991

Characterization of two neuroblastoma cell lines expressing recombinant nerve growth factor receptors.

In earlier studies, a 75,000-dalton glycoprotein (gp75) has been identified as a component of both low- and high-affinity nerve growth factor (NGF) receptors (NGFRs). Using an amphoteric expression vector, we have introduced the cDNA encoding the human gp75 into two neuroblastoma cell lines. SHEP is a human neuroblastoma cell line that lacks most neuronal characteristics and does not express NGFRs. The transformant line SHEP/NGFR expressed a single affinity class of NGF binding sites, did not display NGF-induced up-regulation of fos oncogene expression, and did not efficiently internalize NGF. LAN5 is a neuroblastoma cell line with neuronal characteristics, including expression of neurofilament and display of short neurites. This cell line expresses a small number of high-affinity NGFRs but no detectable low-affinity sites. The transformant line LAN5/NGFR expressed both high- and low-affinity NGFRs, displayed NGF-induced up-regulation of fos oncogene, and efficiently internalized NGF. The number of high-affinity NGF binding sites was nearly the same for LAN5 and LAN5/NGFR, a finding suggesting that there is a limiting number of some separately coded factor or subunit that is required for high-affinity NGFRs. Because NGF induction of fos oncogene expression correlated with expression of high-affinity NGFRs, the putative second factor may also limit NGF responsiveness.

Binding Sites↗
PubMed1990

Growth factor-dependent differentiation along the myeloid and lymphoid lineages in an immature acute T lymphocytic leukemia.

Bone marrow cells from a child with an immature (CD2+, CD5+, CD7+) acute T lymphocytic leukemia (T-ALL) were cultured in the presence and absence of human rIL-2, IL-3, or granulocyte-macrophage (GM)-CSF. Cells cultured without growth factors failed to divide and those initiated in the presence of IL-2 or GM-CSF underwent maturation and terminal T lymphoid or myelomonocytic differentiation, respectively. In contrast, a permanent growth factor-dependent cell line, designated TALL-103/3, was established upon culture in IL-3. The TALL-103/3 cells gradually lost the T cell-specific markers and acquired a myeloid phenotype (CD15+, CD33+). Switching of the IL-3-dependent cells at an early passage to medium containing only human rIL-2 resulted in the establishment of a subline, named TALL-103/2, with a T lymphoid phenotype (CD3+, CD8+, TCR-gamma delta +, CD7+). The TALL-103/2 cells strictly require IL-2 for growth, are irreversibly committed to the lymphoid lineage, and cannot survive in the presence of any other hemopoietic growth factor tested so far. In contrast, the IL-3-dependent TALL-103/3 cells could be adapted to grow in synthetic (serum-free) medium also in the presence of either GM-CSF or IL-5, in which they retain a myeloid phenotype. Interestingly, after 18 mo in culture in IL-3, the TALL-103/3 cells can still be phenotypically converted to the lymphoid lineage upon addition of IL-2, thus maintaining its bipotentiality. Despite the marked phenotypic differences, the TALL-103/2 and TALL-103/3 cell lines show the same karyotypes with multiple abnormalities present in the primary malignant clone and have identical rearrangements of the TCR-gamma and -delta loci, thus confirming their derivation from a common precursor cell. Together, these findings indicate that the phenotype of immature T-ALL cells can be drastically modified by the presence of specific hemopoietic growth factors in the environment, leading to either lymphoid or myeloid lineage commitment while leaving their karyotype and genotype intact.

Antigens, CD↗