Biomedical subjects
Publications and source records attributed to C G Begley.
G-CSF administration leads to significant elevation in the levels of circulating progenitor cells. Infusion of these cells after high-dose chemotherapy is associated with accelerated platelet engraftment that has a considerable impact in shortening thrombocytopenia and reducing need for platelet transfusions.
The SCL gene encodes a putative transcription factor with a basic helix-loop-helix (B-HLH) motif and is known to be predominantly expressed in erythroid cells. Here we also demonstrate expression of SCL mRNA in normal mast cells, mast cell lines and megakaryocytic cell lines. SCL is therefore expressed in the same three lineages as GATA-1, a well-recognized hemopoietic transcription factor. SCL and GATA-1 mRNA were also co-expressed in interleukin 3-dependent primitive myeloid lines. In murine erythroleukemia (MEL) cells SCL and GATA-1 underwent coordinated biphasic modulation during hexamethylene bisacetamide (HMBA)-induced erythroid differentiation. The kinetics of SCL and GATA-1 mRNA expression was inversely correlated with changes in ID, a negative regulator of B-HLH proteins, and was distinct from changes in MYC, MYB and erythropoietin receptor transcripts. During myeloid differentiation of K562 cells, SCL and GATA-1 mRNA levels also underwent biphasic modulation. Thus SCL and GATA-1 are coordinately expressed in multiple hemopoietic lineages and coordinately regulated during induced erythroid and myeloid differentiation. In nonhemopoietic tissues SCL was only detected in adult and developing brain where GATA-1 is reportedly not expressed. In day 14.5 embryos analysed by in situ hybridization, SCL transcripts were detected in post-mitotic neurons in the metencephalon and roof of the mesencephalon. This suggests a previously unexpected role for SCL in neural differentiation.
The alpha-chain of the interleukin 2 receptor (IL-2R alpha) is expressed on monocytes and macrophages after activation by bacterial lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma). In the present study, we investigated whether the expression of IL-2R alpha is associated with the process of differentiation of myeloid cells to mature macrophages and how this expression is regulated. The murine myeloid M1 cell line, which can be induced by leukemia inhibitory factor (LIF) or interleukin 6 (IL-6) to differentiate from blast cells to mature macrophages, was used as a model system for myeloid differentiation. Bone marrow (BM)-derived macrophages were used as mature myeloid cells. Cytofluorometry revealed that IL-2R alpha is transiently expressed during M1 cell differentiation, with peak levels 24 h after induction by LIF or IL-6, whereas the high affinity receptor for monomeric IgG2a (FcR), a surface marker typical for macrophage differentiation, continues to rise up to 72 h. BM-derived macrophages already express FcR but not IL-2R alpha. IL-2R alpha expression is induced on these cells after treatment by IL-6 for up to 48 h. Treatment of IL-6-induced M1 cells with indomethacin permitted a sustained expression of IL-2R alpha beyond 24 h, and this effect was reversed by the addition of prostaglandin E2 (PGE2). Northern analysis showed that in M1 cells the expression of mRNA for IL-2R alpha, but not for IL-2R beta, is also transient, indicating that cell surface expression of IL-2R alpha is regulated at the mRNA level. These data show that inducers of macrophage differentiation such as LIF and IL-6 can induce a transient expression of the IL-2R alpha-chain in differentiating murine myeloid M1 cells and that autocrine production of PGE2 is involved in the control of the transient expression of this receptor. However, induction of expression of IL-2R alpha by IL-6 appears to be independent of differentiation because it can be induced on fully differentiated BM-derived macrophages as well.
The SCL gene encodes a member of the helix-loop-helix (HLH) family of transcription factors and is reportedly involved in up to 25% of T-cell acute lymphoblastic leukemia (T-ALL). We have surveyed over 120 primary human tumors including melanomas, myeloid, and lymphoid leukemias, and other solid tumors without evidence of rearrangements involving SCL. These results are further supported by low level expression of SCL in these tumors (as assessed by a polymerase chain-reaction-based method). We conclude that rearrangement/translocation with subsequent activation of SCL occurs infrequently in myeloid leukemias and melanomas.
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The human SCL gene is a member of the family of genes that encode the helix-loop-helix (HLH) class of DNA-binding proteins. A murine SCL cDNA was isolated from a normal macrophage cDNA library by using HLH-specific oligonucleotides as hybridization probes. The coding region is 987 base pairs and encodes a predicted protein of 34 kDa. The nucleotide sequence of the coding region shows 88% identity to the human SCL gene, and the amino acid sequence is 94% identical. The HLH motif and upstream hydrophilic region are entirely conserved in the murine and human proteins. The identity between the mouse and human sequences was less marked in the 5' and 3' untranslated regions. Two murine SCL transcripts that differ in the 3' noncoding region have been detected in fetal liver and various cell lines. Variation was also observed in the 5' untranslated region. Interestingly, immediately downstream of the protein-termination codon, both the human SCL sequence and the murine homolog share an E-box element--the suggested target site for DNA binding of HLH proteins. The murine SCL homolog was mapped to the central part of chromosome 4.
The SCL gene encodes a member of the helix-loop-helix family of transcription factors that have been implicated in regulation of differentiation and development. Although SCL mRNA is not detectable in normal thymocytes or peripheral T-lymphocytes, transcriptional activation occurs in T-cell tumours. A clue to the normal function of SCL has come from demonstration of high levels of SCL mRNA in erythroid cells. To illuminate the function of SCL in the erythroid lineage, an antisense SCL construct was introduced into the human erythroleukaemia cell line, K562. Cells electroporated with a vector containing antisense SCL grew more slowly than control cells which had received vector alone. Non-specific toxicity was excluded by showing that antisense SCL did not influence growth of Raji cells, a B-cell line that does not express endogenous SCL mRNA. Suppression of K562 growth was accompanied by increased spontaneous erythroid differentiation as measured by benzidine staining. K562 cells containing antisense SCL produced smaller colonies in agar and exhibited reduced clonogenicity compared with control cells. In addition, experiments in which K562 colonies were recloned showed that antisense SCL profoundly suppressed self-renewal of K562 cells. These data provide the first evidence that SCL promotes self-renewal in an erythroid cell line and raise the possibility that SCL may function to regulate proliferation of normal erythroid cells.
A new class of DNA-binding proteins with a helix-loop-helix (HLH) structure has recently been described. Many of these transcriptional regulators are known to play a central role in cell-specification and differentiation processes. Four members of the HLH family are now implicated in the development of human lymphoid malignancies as a result of aberrant expression following chromosomal translocation events. This review focuses on two of these family members: SCL and LYL-1.
This investigation was designed to compare the effects of three rigid gas permeable (RGP) contact lens solutions on the rabbit corneal epithelium. Boston Advance Conditioning Solution, Boston Conditioning Solution, and Allergan Wet-N-Soak, which are preserved with 0.0015% polyaminopropyl biguanide, 0.006% chlorhexidine gluconate, and 0.003% benzalkonium chloride, respectively, were evaluated by scanning (SEM) and transmission electron microscopy (TEM). Our results show that Boston Advance Conditioning Solution is significantly more toxic to the corneal epithelium than either Boston Conditioning Solution or Allergan Wet-N-Soak Plus. This is presumably due to the presence and concentration of the preservative, 0.0015% polyaminopropyl biguanide. Although this study was conducted using rabbits, the results raise clinical concerns for human RGP contact lens wearers.
Trabecular meshwork cells from an inbred strain of mice (A/J) were established in tissue culture. Within 1 hour of enucleation, tissue containing the cornea and the chamber angle was excised and placed in tissue culture. Two to five days later, three cell types grew from the explants. Two of these cell types, corneal endothelium and fibroblasts, grew together, with the fibroblasts preferentially spreading on top of the endothelial cells. The trabecular meshwork cells extended from the explant as a distinct morphological type. The corneal endothelium and its associated fibroblasts were then removed from the culture flask with a sterile cotton swab, leaving a monolayer of pure trabecular meshwork cells. These cells required 3-4 weeks to reach confluency and could be passaged five times. They were actively phagocytic in culture and exhibited immunoreactivity to antibodies against two extracellular matrix components, laminin and collagen type IV. Mouse trabecular meshwork cells also expressed receptors for acetylated low-density lipoprotein, a property shared by trabecular meshwork cells derived from other species. The availability of trabecular meshwork cells from an inbred strain of mice will facilitate future in vivo functional studies of these cells in a syngeneic system, as well as investigations of potential immunoregulatory properties of the trabecular meshwork.
Approximately 6.8 percent of soft contact lens wearers develop multiple nodular deposits on the front surface of their soft contact lenses. It was the purpose of this investigation to evaluate the role of calcium in the newly formed and mature deposits. Nodular deposits were examined using scanning electron microscopy and Energy Dispersive X-ray (EDX) analysis for calcium content. Any deposit which did not demonstrate the presence of calcium was sectioned, and the individual section re-examined by EDX analysis. Our results indicate that calcium was present in all but three of 72 nodular deposits investigated. The calcium was finely distributed throughout the deposits in a non-crystalline pattern, especially in the basal layers. Sections of the deposits also were examined at the light microscope level for the presence of lipids, calcium, and polysaccharides (mucin). All deposits stained positively for lipids, but polysaccharides were more evident in newer deposits. These results may indicate that both calcium and polysaccharides are involved in the genesis of nodular deposits.
The helix-loop-helix genes LYL, SCL and E2A are associated with chromosome translocations found in human lymphoid leukemias. To establish their hematopoietic expression patterns, we have isolated murine LYL and SCL cDNA clones and investigated the expression of all three genes by Northern blot analysis of 58 murine hemopoietic cell lines and tissues. The nucleotide sequences of LYL cDNA clones revealed alternative 5' untranslated sequences and differential splicing within the 5' portion of the coding region that may produce a LYL polypeptide lacking an N-terminal segment. The LYL gene was expressed in most myeloid, erythroid and B lymphocyte cell lines and displayed two alternative size classes of transcripts, the smaller size class (1.5-1.8 kb) being typical of the erythroid lineage and the larger class (2.0-2.3 kb) of the B cell lineage. These two size classes were found to differ in the 5' untranslated region. Thus, expression of the LYL gene appears to be differentially regulated in different hemopoietic cell types. In contrast, the E2A gene was expressed throughout the hemopoietic compartment as a single dominant transcript (3.5 kb). SCL expression was restricted to erythroid, mast and early myeloid cell lines, and the level of SCL transcripts (3.0 and 4.7 kb species) increased markedly during DMSO-induced differentiation of erythro-leukemia cells. Hence the SCL gene product may be an important regulatory factor for the erythroid lineage. The low or undetectable expression of both SCL and LYL in most T lymphoid cell sources is consistent with the view that the translocations of these genes in human T cell leukemias alter their normal regulation and may thereby contribute to neoplasia.
The SCL gene encodes a member of the 'helix-loop-helix' family of DNA binding regulatory proteins. It is transcriptionally activated in some cases of T-cell acute lymphoblastic leukaemia by a reciprocal translocation involving the T cell receptor delta locus. In order to gain insight into the normal functions of SCL we have studied SCL mRNA levels in human and murine haemopoietic cell lines and normal tissues. We have observed high levels of SCL mRNA in all human and murine erythroid cell lines examined. Foetal and adult normal haemopoietic cell populations rich in erythroid precursors also expressed high levels of SCL mRNA. Our results suggest a previously unexpected role for SCL in the regulation of erythropoiesis.
The nature of the interleukin 2 (IL-2) receptor on purified human B lymphocytes was examined. Both normal and malignant cells showed evidence of a 70-75,000 mol. wt (p75) IL-2 binding molecule as assessed by 125I-labeled IL-2 binding and receptor cross-linking. On normal, Tac-negative B lymphocytes the estimated number of p75 binding sites was 1100 per cell and the dissociation constant (Kd) was 1.7 nM. Consistent with this, cross-linking experiments demonstrated the presence of an IL-2 binding molecule of 70-75,000 mol. wt. Purified B cells from patients with hairy cell leukemia and chronic lymphocytic leukemia (CLL) also expressed the p75 IL-2 binding molecule. In the HCL samples, a small number of high-affinity IL-2 binding sites were detected (27-90) while the majority of binding sites (2100-10,800) were typical of low-affinity p55 Tac binding. IL-2 added to the purified normal and CLL B lymphocytes led to the induction of p55 Tac expression and the generation of high-affinity IL-2 receptors. This response to IL-2 was equivalent to the response observed when normal B lymphocytes were stimulated by Staphylococcus aureus Cowan I.
Giant papillary conjunctivitis (GPC) is largely a soft contact lens-related syndrome, characterized by the formation of giant papillae on the upper tarsal conjunctiva, itching, excess mucus, erythema, and contact lens intolerance. In response to the suggestion that GPC occurs more frequently in atopic individuals, a retrospective study was designed in order to determine the months in which patients were diagnosed with GPC during 1987 and 1988. Personal histories of allergy were also recorded for these patients, and compared to an age- and sex-matched control group. Significant peaks in the number of patients diagnosed with GPC occurred in the spring, and especially in late summer/early fall of both years. In addition, the GPC patients reported significantly more overall allergies than did the control group. However, a breakdown of the individual allergy data revealed that only allergies to contact lens solutions, specifically thimerosal, were significantly higher in the GPC group. Reported allergies to medications and pollen were also elevated in the GPC patients, but not significantly. The seasonal onset of GPC diagnoses in 1987 and 1988, and the increase in reported allergies within the GPC group, suggests a strong association between atopy and the development of GPC.
We describe the structural organization of the human SCL gene, a helix-loop-helix family member which we believe plays a fundamental role in hematopoietic differentiation. The SCL locus is composed of eight exons distributed over 16 kb. SCL shows a pattern of expression quite restricted to early hematopoietic tissues, although in malignant states expression of the gene may be somewhat extended into later developmental stages. A detailed analysis of the transcript(s) arising from the SCL locus revealed that (i) the 5' noncoding portion of the SCL transcript, which resides within a CpG island, has a complex pattern of alternative exon utilization as well as two distinct transcription initiation sites; (ii) the 5' portions of the SCL transcript contain features that suggest a possible regulatory role for these segments; (iii) the pattern of utilization of the 5' exons is cell lineage dependent; and (iv) all of the currently studied chromosomal aberrations that affect the SCL locus either structurally or functionally eliminate the normal 5' transcription initiation sites. These data suggest that the SCL gene, and specifically its 5' region, may be a target for regulatory interactions during early hematopoietic development.
Immunoglobulin (Ig) and T-cell receptor (TCR) genes were examined in the lymphoblasts of 70 children with immunophenotypically defined B-cell precursor acute lymphoblastic leukemia (ALL). The most frequent genes to rearrange were Ig heavy (H) chain (93%) and TCR delta (79%), followed by TCR gamma (49%), Ig kappa and/or lambda light (L) chain (46%), TCR alpha (46%), and TCR beta (29%). Thus, despite their putative "B-cell precursor" lineage, these leukemias manifest a remarkably high incidence of TCR gene rearrangements. While certain patterns predominate, there is considerable heterogeneity in Ig and TCR genotypes in this disease. No significant associations were found between Ig and TCR genotype and commonly used prognostic factors including age, sex, race, WBC, French-American-British (FAB) subtype, or cytogenetics. However, the lymphoblasts of three of six patients who failed to achieve initial remission had germline patterns of every Ig and TCR gene, a genotype not observed in the leukemic cells from any of the 64 patients who achieved complete remission (p2 = .0007). This study suggests that particular Ig and TCR genotypes may be of clinical relevance in childhood B-cell precursor ALL. The finding of rearranged TCR genes in a large proportion of cases raises fundamental questions about early lineage commitment and lymphocyte differentiation along B-cell and T-cell pathways.
Currently, in the United States, the Food and Drug Administration has approved a number of enzyme cleaners for use with soft contact lenses. These cleaners contain three active enzymes--papain, pancreatin, and subtilisin. Although several studies have compared the cleaning efficacy of papain and pancreatin, little information exists concerning subtilisin. It was the aim of this investigation to compare the performances of four marketed enzyme cleaners, including Allergan Enzymatic (papain), Optizyme (pancreatin), ReNu Effervescent (subtilisin), and Ultrazyme (subtilisin A). Lenses were examined using the scanning electron microscope, and the amount of deposits on the lens surfaces quantified by digital analysis. All of the enzyme cleaners were effective in removing deposits from the contact lenses surfaces, but there was no significant difference in the cleaning efficacy of the four cleaners tested.