Survival of leukemic B cells promoted by engagement of the antigen receptor.

Chronic lymphocytic leukemia (CLL) is an incurable leukemia characterized by the slow but progressive accumulation of cells in a CD5+ B-cell clone. Like the nonmalignant counterparts, B-1 cells, CLL cells often express surface immunoglobulin with the capacity to bind autologous structures. Previously there has been no established link between antigen-receptor binding and inhibition of apoptosis in CLL. In this work, using primary CLL cells from untreated patients with this disease, it is demonstrated that engagement of surface IgM elicits a powerful survival program. The response includes inhibition of caspase activity, activation of NF-kappaB, and expression of mcl-1, bcl-2, and bfl-1 in the tumor cells. Blocking phosphatidylinositol 3-kinase (PI3-K), a critical mediator of signals through the antigen receptor, completely abrogated mcl-1 induction and impaired survival in the stimulated cells. These data support the contention that CLL cell survival is promoted by antigen for which the malignant clone has affinity, and suggest that pharmacologic interference with antigen-receptor-derived signals has potential for therapy in patients with CLL.

dCtBP-dependent and -independent repression activities of the Drosophila Knirps protein.

Transcriptional repressor proteins play essential roles in controlling the correct temporal and spatial patterns of gene expression in Drosophila melanogaster embryogenesis. Repressors such as Knirps, Krüppel, and Snail mediate short-range repression and interact with the dCtBP corepressor. The mechanism by which short-range repressors block transcription is not well understood; therefore, we have undertaken a detailed structure-function analysis of the Knirps protein. To provide a physiological setting for measurement of repression, the activities of endogenous or chimeric Knirps repressor proteins were assayed on integrated reporter genes in transgenic embryos. Two distinct repression functions were identified in Knirps. One repression activity depends on dCtBP binding, and this function maps to a C-terminal region of Knirps that contains a dCtBP binding motif. In addition, an N-terminal region was identified that represses in a CtBP mutant background and does not bind to the dCtBP protein in vitro. Although the dCtBP protein is important for Knirps activity on some genes, one endogenous target of the Knirps protein, the even-skipped stripe 3 enhancer, is not derepressed in a CtBP mutant. These results indicate that Knirps can utilize two different pathways to mediate transcriptional repression and suggest that the phenomenon of short-range repression may be a combination of independent activities.

Inhibition of NF-kappaB induces apoptosis of KSHV-infected primary effusion lymphoma cells.

Kaposi sarcoma-associated herpesvirus (KSHV), or human herpervirus 8 (HHV-8), is a gamma-herpesvirus that infects human lymphocytes and is associated with primary effusion lymphoma (PEL). Currently, the role of viral infection in the transformation of PEL cells is unknown. One possibility is that KSHV, like the lymphotropic viruses Epstein-Barr virus (EBV) and human T-cell leukemia virus I (HTLV-I), activates the transcription factor NF-kappaB to promote survival and proliferation of infected lymphocytes. To examine this possibility, we assessed NF-kappaB activity in KSHV-infected PEL cell lines and primary tumor specimens by electrophoretic mobility shift assay (EMSA). We observed that NF-kappaB is constitutively activated in all KSHV-infected lymphomas, and consists of 2 predominant complexes, p65/p50 heterodimers and p50/p50 homodimers. Inhibition experiments demonstrated that Bay 11-7082, an irreversible inhibitor of IkappaBalpha phosphorylation, completely and specifically abrogated the NF-kappaB/DNA binding in PEL cells. PEL cells treated with Bay 11 demonstrated down-regulation of the NF-kappaB inducible cytokine interleukin 6 (IL-6), and apoptosis. These results suggest that NF-kappaB activity is necessary for survival of KSHV-infected lymphoma cells, and that pharmacologic inhibition of NF-kappaB may be an effective treatment for PEL.

Recovery from the lethal effects of saxitoxin: a therapeutic window for 4-aminopyridine (4-AP).

We have shown that saxitoxin (STX) induced lethality can be reversed by 4-AP when it is administered at the time of respiratory arrest [Benton, B. J., Spriggs, D. L., Capacio, B. R. and Chang, F.-C. T. (1995) 4-Aminopyridine antagonizes the lethal effects of saxitoxin (STX) and tetrodotoxin (TTX). International Society of Toxicology, 5th Pan American Symposium on Animal, Plant and Microbial Toxins, Frederick, MD. July/August 1995, p. 217]. The purpose of this study was to determine whether 4-AP's efficacy could be enhanced further when administered at different times relative to STX intoxication. The animals used in this study were chronically instrumented for concurrent recordings of diaphragm electromyogram (DEMG), neck skeletal muscle electromyogram, Lead II electrocardiogram, and electrocorticogram (ECoG). There were five groups of unanesthetized guinea pigs. The first group served as 4-AP controls and received a 2 mg/kg i.m. dose of 4-AP. The four remaining groups were given a lethal dose of STX (5 microg/kg i.m.); the second group, STX controls, received no 4-AP; the third group, the 4-AP treatment group, received 4-AP immediately following cardiorespiratory collapse; the fourth group was the 4-AP/STX co-administration group and 4-AP was given concurrently with STX; and the fifth group was the 4-AP pretreatment group in which 4-AP was given 10 min before STX. At the point of STX-induced cardiorespiratory collapse, the guinea pigs were ventilated and given an i.p. injection of sodium bicarbonate. Results showed that 4-AP prevented cardiorespiratory collapse in 3/7 animals in the 4-AP pretreatment group. Also, 4-AP in conjunction with artificial ventilation and sodium bicarbonate accelerated recovery from STX-induced cardiorespiratory collapse in all the treatment groups compared to the STX controls.

4-Aminopyridine reverses saxitoxin (STX)- and tetrodotoxin (TTX)-induced cardiorespiratory depression in chronically instrumented guinea pigs.

The extent to which cardiorespiratory infirmity and other sublethal effects of saxitoxin (STX) and tetrodotoxin (TTX) can be reversed by 4-aminopyridine (4-AP) was investigated in guinea pigs chronically instrumented for the concurrent electrophysiological recordings of electrocorticogram (ECoG), diaphragmatic electromyogram (DEMG), Lead II electrocardiogram, and neck skeletal muscle electromyogram. Animals were intoxicated with either STX or TTX (2 and 3 microg/kg, im) to produce a state of progressive cardiorespiratory depression (depicted by decreasing DEMG amplitude, bradypnea, and bradycardia). At the point where cardiorespiratory performance was most seriously compromised (approximately 30 min posttoxin), 4-AP (1 or 2 mg/kg, im) was administered. The therapeutic effect of 4-AP was striking in that, within minutes, the toxin-induced diaphragmatic blockade, bradypnea, bradycardia, and depressed cortical activity were all restored to a level either comparable to, or surpassing, that of control. The optimal 4-AP dose level was determined to be 2 mg/kg (im) based on analyses of cardiorespiratory activity profiles throughout the course of intoxication and 4-AP treatment. At the dose levels (either 1 or 2 mg/kg) used to restore ventilatory function and cardiovascular performance, 4-AP produced no sign of seizures and convulsions. Although less serious secondary effects such as cortical excitant/arousal effect (indicated by ECoG power spectral analysis) and transient periods of skeletal muscle fasciculation were observed, these events were of minor concern particularly in view of the remarkable therapeutic effects of 4-AP.

Electrophysiology and density of retinal neurons in mice with a mutation that includes the Pax2 locus.

PURPOSE: The Krd mouse has a deletion in chromosome 19 that includes the Pax2 gene locus. The aim of this study was to characterize in detail how these retinas differ from normal. METHODS: Both electroretinographic and anatomic methods were used to assess visual function. Full-field flash electroretinograms (ERGs) and planimetric densities were obtained from Krd and control animals. RESULTS: Measurements of the ERG show that in the Krd mice, both a- and b-wave amplitudes are attenuated relative to control by amounts that vary from animal to animal. The b-wave of the ERG generally is affected more severely than the a-wave. However, there is little or no shift of the curves relating the b-wave and a-wave amplitude to the intensity of the stimulus. Also, no change in the response kinetics seems to be associated with the attenuated responses. Estimates of planimetric cell density in the outer nuclear, inner nuclear, and ganglion cell layers show significant cell losses in affected animals that are more pronounced proportionally in the inner layers. Comparisons between electrophysiological and histologic measurements made on each eye show good correlation between the reduction in the ERG components and the magnitude of cell losses. CONCLUSIONS: These experiments show that the eyes of Krd mice have reduced ERGs and reduced cellular density. There is a loss of cells in all layers of the retina, but the inner layers are affected more severely. Consistent with this, the b-wave is reduced more than the a-wave. The normal functional dependency of the ERG on stimulus intensity and the normal response kinetics suggest the cellular losses are not associated with changes in cellular function.

4-Aminopyridine antagonizes saxitoxin-and tetrodotoxin-induced cardiorespiratory depression.

Antagonism of saxitoxin-and tetrodotoxin-induced lethality by 4-aminopyridine was studied in urethane-anesthetized guinea pigs instrumented for the concurrent recordings of medullary respiratory-related unit activities (Bötzinger complex and Nu. para-Ambiguus), diaphragmatic electromyogram, electrocorticogram, Lead II electrocardiogram, blood pressure, end-tidal CO2 and arterial O2/CO2/pH. The toxin (either saxitoxin or tetrodotoxin) was infused at a dose rate of 0.3 microgram/kg/min (i.v.) to produce a state of progressive cardiorespiratory depression. The animals were artificially ventilated when the magnitude of integrated diaphragm activities was reduced to 50% of control. Immediately after the disappearance of the diaphragm electromyogram, the toxin infusion was terminated, and 4-aminopyridine (2 mg/kg, i.v.) was administered. The therapeutic effect of 4-aminopyridine was striking in that the toxin-induced blockade of diaphragmatic neurotransmission, vascular hypotension, myocardial anomalies, bradycardia and aberrant discharge patterns of medullary respiratory-related neurons could all be promptly restored to a level comparable to that of control condition. The animals were typically able to breathe spontaneously within minutes after 4-aminopyridine. At the dose level used to achieve the desired therapeutic responses, 4-aminopyridine produced no sign of seizure and convulsion. Although less serious side-effects such as cortical excitant/arousal and transient periods of fascicular twitch could be observed, these events were of minor concern, in our opinion, particularly in view of the remarkable therapeutic effects of 4-aminopyridine.

Kidney and retinal defects (Krd), a transgene-induced mutation with a deletion of mouse chromosome 19 that includes the Pax2 locus.

The semidominant mutation Krd (kidney and retinal defects) was identified in transgenic line Tg8052. Krd/+ mice have a high incidence of kidney defects including aplastic, hypoplastic, and cystic kidneys. Retinal defects in Krd/+ mice include abnormal electroretinograms and a reduction of cell numbers that is most extreme in the inner cell and ganglion layers. Viability of Krd/+ mice is strongly influenced by genetic background, and growth retardation is observed in young animals. Homozygosity results in early embryonic lethality. Fluorescence in situ hybridization of a transgene-specific probe localized the insertion site to the distal region of mouse Chromosome 19. The sequence of the insertion site revealed transgene insertion into a LINE element with deletion of a single nucleotide from the 3' terminus of the transgene. A polymorphic microsatellite, D19Umi1, was identified in a junction clone and mapped in several large crosses. D19Umi1 is located 1.7 +/- 1.0 cM distal to Pax2, which encodes a paired type transcription factor expressed in embryonic kidney and eye. Deletion of Pax2 from the transgenic chromosome was demonstrated by Southern analysis of genomic DNA from (Krd/+ x SPRET/Ei)F1 mice. Additional genetic and molecular data are consistent with an approximately 7-cM deletion that includes the loci stearoyl CoA desaturase (Scd1), pale ear (ep), D19Mit17, D19Mit24, D19Mit27, D19Mit11, and Pax2. This deletion, Del(19)TgN8052Mm, will be useful for genetic and functional studies of this region of mouse Chromosome 19.

Molecular map of chromosome 19 including three genes affecting bleeding time: ep, ru, and bm.

The mouse ruby eye (ru) and pale ear (ep) pigment dilution genes cause platelet storage pool deficiency (SPD) and prolonged bleeding times. The brachymorphic (bm) gene, in addition to causing skeletal abnormalities, is also associated with prolonged bleeding times. All three hemorrhagic genes are found within 10 cM on Chromosome (Chr) 19. In this study, 15 microsatellite markers and five cDNAs, spanning 21 cM of Chr 19, were mapped in relation to the bm, ep, and ru genes in 457 progeny of an interspecific backcross utilizing the highly inbred strain PWK derived from the Mus musculus musculus species. Several markers were found to be closely linked to the three genes and should be useful as entry points in their eventual molecular identification.

Insertional mutation of the hairless locus on mouse chromosome 14.

Crosses between heterozygous transgenic mice from line 5053 produced offspring with progressive irreversible hair loss beginning at day 19. With increasing age, the skin of these animals became thicker and plicated in appearance. Histological analysis revealed the complete absence of normal hair follicles and numerous intradermic cystic structures, which enlarged with time and became filled with keratinaceous material. Test crosses demonstrated that the affected animals are homozygous for the transgene insertion. The clinical and histological phenotype of the new mutant closely resembles that of the rhino allele at the hairless locus on Chromosome (Chr) 14. Complementation tests and linkage analysis indicate that the transgene has interrupted the hairless locus. It has been demonstrated previously that mutation at the hr locus is accompanied by a variety of immune deficiencies. Many of the older affected transgenic mice developed an impetigo-like skin eruption which responded to antibiotic ointment and which may reflect impaired immune function. The transgenic allele, hrTgN5053Mm, will be useful for identification of the transcription unit of the hairless locus.

Transgene-induced mutation of the murine steel locus.

The product of the steel locus is essential for normal development of three distinct populations of stem cells--the neural crest-derived melanoblasts, germ cells, and blood cell precursors. Many mutant alleles at steel are lethal in homozygotes and produce coat color dilution in heterozygotes. We have identified a transgenic mouse with diluted pigmentation that closely resembles that of steel heterozygotes. We have demonstrated that the site of transgene insertion is genetically linked to the phenylalanine hydroxylase locus on mouse chromosome 10. In addition, the chromosome carrying the transgene fails to complement the recessive lethality of the Sl allele of steel and the pigmentation defect of the Slpan allele. The data indicate that the inserted transgene has disrupted the steel locus. The resulting allele, designated Sltg, provides a molecular tag for isolation of the steel gene, as well as a new allele for characterization of this developmentally important locus.

Regulation of amylase gene expression in diabetic mice is mediated by a cis-acting upstream element close to the pancreas-specific enhancer.

We localized the cis-acting sequences that mediate the regulation of a pancreatic amylase gene, Amy-2.2, in diabetic mice. We constructed three hybrid genes containing sequences from the 5'-flanking region of the amylase gene upstream of the heterologous elastase promoter linked to the CAT structural gene. These constructs were transferred to the germ line of transgenic mice by microinjection of fertilized eggs. The amylase sequences had two effects on expression of the elastase promoter: Basal expression was increased, and expression in diabetic animals was reduced to approximately 2% of basal levels. A 30-bp amylase fragment was sufficient to transfer both of these regulatory functions to the elastase promoter. Sequences within this 30-bp fragment are included in the binding site for the pancreatic nuclear protein PTF1. The close association of the PTF1-binding site and the regulatory functions is consistent with a mechanism based on interference with activation by PTF1 in diabetic pancreas. PTF1-binding activity is not reduced in diabetic pancreas. The data presented here demonstrate that the 5'-flanking region of the pancreatic amylase gene contains a novel insulin-dependent element (IDE) that mediates the loss of expression in diabetic animals.

A salivary amylase transgene is efficiently expressed in liver but not in parotid gland of transgenic mice.

Two distinct mouse amylase cDNAs, corresponding to the genes Amy-1.1 and Amy-1.2, have been isolated from a YBR/Ki parotid cDNA library. A cosmid clone containing the intact Amy-1.1 gene from strain YBR/Ki, including both the parotid and liver promoters, was transferred to the germ line of C57BL/6J mice. Two independent transgenic lines were produced. The transferred genes are organized as a 4-copy autosomal locus in line Tg518 and an 8-copy Y-linked locus in line Tg2736. Serum of both transgenic lines contained high levels of the AMY1B isozyme encoded by the transferred gene. Transcripts were detected in liver and, at a lower level, in several other tissues including white and brown fat. The anticipated expression in parotid was not observed. Constructs containing 270 or 540 bp of the 5' flanking region of the parotid promoter, cloned upstream of the chloramphenicol acetyltransferase (CAT) structural gene, were also not expressed in transgenic mice. The results suggest that sequences located more than 5 kb upstream of the Amy-1 parotid promoter and/or more than 10 kb downstream from the structural gene are required for parotid-specific expression. The results also demonstrate that non-parotid sources can produce a normal level of AMY1 in serum. Liver is the probable source of AMY1 in serum of these transgenic mice.

Insulin response of a hybrid amylase/CAT gene in transgenic mice.

Expression of an amylase/CAT hybrid gene was analyzed in transgenic mice. The amylase promoter was derived from a pancreatic amylase gene whose expression is repressed in diabetic animals. Pancreas-specific expression of the amylase/chloramphenicol acetyl-transferase (CAT) construct was observed in two independent transgenic lines. Correct initiation of transcription was demonstrated by protection of an anti-sense riboprobe. To evaluate the insulin dependence of the hybrid gene, diabetes was induced by treatment with streptozotocin. As a result of this treatment, pancreatic CAT activity was reduced to undetectable levels. Subsequent administration of insulin restored CAT activity to normal levels. The abundance of CAT transcripts was also greatly reduced in diabetic pancreas. These studies localize the determinants of pancreas specificity and insulin dependence to the region between -208 and +19 of the mouse pancreatic Amy-2.2 gene. The results are consistent with an effect of insulin on amylase transcription, rather than post-transcriptional regulation of mRNA processing or stability.

Tissue-specific and insulin-dependent expression of a pancreatic amylase gene in transgenic mice.

The regulatory properties of mouse pancreatic amylase genes include exclusive expression in the acinar cells of the pancreas and dependence on insulin and glucocorticoids for maximal expression. We have characterized a murine pancreatic amylase gene, Amy-2.2y, whose promoter sequence is 30% divergent from those of previously sequenced amylase genes. To localize sequences required for tissue-specific and hormone-dependent activation, we established two lines of transgenic mice. The first line contained a single copy of the complete Amy-2.2y gene as well as 9 kilobases of 5'-flanking sequence and 5 kilobases of 3'-flanking sequence. The second line carried a minigene which included 208 base pairs of 5'-flanking sequence and 300 base pairs of 3'-flanking sequence. In both lines the transgene was expressed at high levels exclusively in the pancreas. Both constructs were dependent on insulin and induced by dexamethasone. Thus, the transferred genes contained the sequences required for tissue-specific and hormonally regulated expression.