Differential induction of apoptosis and MAP kinase signaling by mitochondrial toxicants in drug-sensitive compared to drug-resistant B-lineage lymphoid cell lines.

A panel of human B-lineage lymphoma cell lines differing in cancer drug-resistance status and Bcl-2/Bax expression were used to study the contribution of mitochondrial-based perturbations and regulation in differential induction of apoptosis. Mitochondrial dysfunction was induced in cells by the uncoupler carbonyl cyanide m-chlorophenylhydrazone (mClCCP) and the respiratory chain inhibitor antimycin A. Cells were then assayed for early changes in MAP kinase signaling and subsequent induction of apoptosis. The cancer drug-resistant cell lines EW36 and CA46, overexpressing Bcl-2 and deficient in Bax, respectively, were both resistant to mitochondrial toxicant-induced cleavage of poly(ADP-ribose) polymerase (PARP) and morphologically detectable apoptotic cell death. In contrast, cancer drug-sensitive ST486 cell line, with low Bcl-2 expression, was sensitive to PARP cleavage and apoptosis engagement. Interestingly, mClCCP induced twofold more apoptosis than antimycin A in the ST486 cells. Exposure to the mitochondrial toxicants resulted in the early and preferential activation of the ERK and p38 MAP kinase pathways in only the drug-sensitive ST486 cell line, with mClCCP more potent than antimycin A. Specific inhibition of the p38 pathway augmented baseline and mClCCP-induced apoptosis. These results show that multi-drug-resistant and -sensitive B-lineage cells are also resistant and sensitive to compounds inducing mitochondrial dysfunction. The differential sensitivity to mitochondrial toxicant effects involved regulation by MAP kinases, since ERK and p38 were found to be preferentially activated only in the drug-sensitive B-lineage cells. Modulation of the p38 signaling pathway altered the sensitivity of cells to mitochondrial stress and may play a more general role in regulating the sensitivity of B-lineage cells to drugs and environmental toxicants.

Chondrocyte necrosis and apoptosis in impact damaged articular cartilage.

A decrease in chondrocyte numbers is one characteristic of osteoarthritic cartilage. This decrease may be the result of apoptosis or other forms of cell death induced by mechanical damage. Furthermore, cell death may contribute to the structural and metabolic changes found in osteoarthritic cartilage. Therefore, we investigated cell viability and the mode of cell death in cartilage subjected to an increasing severity of impact loads expected to cause compositional damage and osteoarthritic-like metabolic alterations. Canine cartilage explants were subjected to cyclic indentation impacts of 5 megapascals at 0.3 Hz for 0, 2, 20, and 120 min and then kept in culture for 2, 4, 48, and 144 h. Cell death was assessed by the TUNEL assay and by uptake of propidium iodide. Viable cells were detected by the ability to metabolize fluorescein diacetate. Nuclear morphology and ultrastructure of the cell were examined using Hoechst 33342 fluorescent staining and transmission electron microscopy (TEM). As controls for necrosis and apoptosis, cartilage was, respectively, frozen and thawed or incubated with mitomycin-C, an apoptosis inducer. In cartilage that had been loaded for 2 h, 32% of the chondrocytes in the loaded core took up propidium iodide within 2 h after loading. Most of these were in the middle to superficial zones and reflected leaky cell membranes usually characteristic of necrosis. Less than 1% of these chondrocytes were positive in the TUNEL assay after 4 h. After additional culture for 2 days, however, the proportion of chondrocytes which were positive in the TUNEL assay reached 73%. A dose dependent response to duration of loading was detected with the TUNEL assay at this time. The TUNEL assay was not specific for apoptosis since 92% of chondrocytes in freeze/thawed cartilage were TUNEL positive. However, some cells with apoptotic bodies and chromatin condensation characteristic of apoptosis were found in the transition zone between necrotic and normal chondrocytes, but not in the superficial and upper zones, in impact damaged cartilage. We concluded that in this study, necrosis occurred first, followed by apoptosis.

Expression of cell death regulatory genes and limited apoptosis induction in avian blastodermal cells.

Apoptosis is a well-established cellular mechanism for selective cell deletion during development. However, little is known about the expression of an apoptotic pathway and its role in determining the relative sensitivity of the early, pre-gastrula, avian embryo to stress-induced cell death. We examined the sensitivity of avian blastodermal cells to engage in apoptosis upon exposure to etoposide, a topoisomerase II-inhibitor that rapidly and efficiently induces apoptosis in many cell types. We found that while the blastodermal cells are capable of engaging in apoptosis, they are highly resistant to such induction with respect to both concentration of drug required and length of exposure, even when compared to a tumor cell line with a known multi-drug resistant phenotype. Additionally, we assessed the expression of several candidate regulatory genes in blastodiscs from infertile eggs (i.e., maternal RNA transcripts), blastodermal cells immediately following oviposition, and various stages of early development up to gastrulation. This analysis revealed that several genes whose products have anti-apoptotic activity, including bcl-2, bcl-xL, hsp70, grp78 and the glutathione S-transferases, are expressed as early as the stage 1 embryo in the newly oviposited egg. These transcripts are also found in the infertile blastodisc, suggesting a role for maternally derived transcripts in the protection of the oocyte and zygote. Significantly, constitutive levels of hsp70 mRNA exceeded those of the other anti-apoptotic genes in the blastodermal cells. These results contribute to an emerging picture of stress resistance at the earliest stages of avian embryo development which involves multiple anti-apoptotic genes that act at different regulatory points in the apoptotic cascade.

Contribution of gene-specific lesions, DNA-replication-associated damage, and subsequent transcriptional inhibition in topoisomerase inhibitor-mediated apoptosis in lymphoma cells.

Lymphoid lineage tumor cells differ widely in their relative sensitivity or resistance to the induction of apoptosis by a variety of chemotherapeutic drugs. We used a model system of virally transformed B- and T-lymphoma cell lines to show that avian T-lymphoma cells are highly resistant, whereas B-lymphoma cells are highly sensitive, to the induction of apoptosis by a wide spectrum of chemotherapeutic drugs that induce different types of lesions in DNA. Among the various drugs examined, the topoisomerase inhibitors, camptothecin, actinomycin D, and etoposide, were the most potent inducers of apoptosis. Examination of the relative contribution of DNA replication and transcriptional inhibition to the differential induction of apoptosis by the topoisomerase inhibitors revealed that the signals initiating the apoptotic response vary, even among compounds with similar cellular targets. Specifically, DNA replication plays a major role in the induction of camptothecin-induced apoptosis, and a lesser role in the induction of apoptosis by etoposide. In contrast, DNA replication is not involved in the induction of apoptosis by actinomycin D. Transcriptional inhibition may provide the major cellular signal for apoptosis induction by this compound. In addition, we determined that the extent of topoisomerase I-cleavable complex inhibition is similar even in genes that are transcribed at different levels and by different RNA polymerases. An overexpressed c-myc gene is no more vulnerable to topoisomerase inhibition than its normally expressed counterpart. In contrast, even under conditions yielding similar amounts of topoisomerase inhibition, rRNA genes are more sensitive to transcriptional inhibition than are the c-myc genes.

Cell death in the avian blastoderm: resistance to stress-induced apoptosis and expression of anti-apoptotic genes.

We investigated the expression of an apoptotic cell death program in blastodermal cells prior to gastrulation and the susceptibility of these cells to stress-induced cell death. A low frequency (3.1%) of apoptotic blastodermal cells was observed in Hoechst 33342-vitally stained cytological preparations of complete blastoderms from unincubated eggs. These cells showed the stereotypic features of apoptosis including a progression of nuclear changes, cell shrinkage and blebbing, and the formation of apoptotic bodies. Prolonged storage of eggs at 12 degrees C induced apoptosis in blastodermal cells (14%). A modest amount of apoptosis (10%) was also induced at the heat shock temperature of 48 degrees C, but not at 45 degrees C. Etoposide and other potent cytotoxic drugs failed to induce apoptosis in the blastodermal cells after 4 h of exposure. Progressively more apoptosis was induced at 8 and 24 h, but it did not exceed 35% of the cells. We detected transcripts for the anti-apoptotic genes bcl-2, bcl-xL, and hsp70. The developmental expression of these genes, especially hsp70, correlated with the delayed and limited stress-induction of apoptosis. These studies reveal the capacity of pre-streak blastodermal cells to engage in apoptosis and their relative resistance to stress conditions. This may be due to the prominent expression of hsp70 and/or multiple cell death genes which primarily antagonize cell death.

Involvement of gene-specific DNA damage and apoptosis in the differential toxicity of mitomycin C analogs towards B-lineage versus T-lineage lymphoma cells.

Avian and mammalian B- and T-lineage lymphocytes display differential sensitivity to a variety of genotoxic agents. Specifically, T-lineage cells show a high degree of resistance to the toxic effects of exposure to chemotherapeutic drugs, whereas B-lineage cells show a high degree of sensitivity. We used a model system consisting of virally transformed B- and T-lymphoma cell lines to further define the cellular and molecular mechanisms responsible for the differential toxicity of two chemotherapeutic drugs that induce DNA-interstrand cross-links to different degrees, mitomycin C (MMC) and its aminodisulfide analog, BMY 25067. Quantification of the number of cross-links introduced in the transcriptionally active ribosomal RNA gene cluster revealed that similar levels of DNA damage were induced in B- and T-lymphoma cell lines. However, B-lymphoma cells were highly sensitive to induction of apoptosis and inhibition of growth compared with the more resistant T-lymphoma cells for both compounds. BMY 25067 induced approximately 2-fold more cross-links in rDNA than did MMC, along with a concurrent enhanced induction of apoptosis in both B- and T-lymphoma cell lines. An analysis of the persistence of DNA lesions over multiple cell cycles revealed that neither B- nor T-lymphoma cells repaired DNA cross-links to a significant extent. These data suggest that differences in the extent or persistence of DNA-interstrand cross-links are not responsible for the differential toxicity of MMC and its analog towards B- versus T-lineage cells. Rather, differential drug toxicity involves early and extensive entry into apoptosis in B-lymphoma cells contrasted to the delayed and minimal apoptotic induction in T-lymphoma cells.

Selective mitomycin C and cyclophosphamide induction of apoptosis in differentiating B lymphocytes compared to T lymphocytes in vivo.

Differentiating B and T lymphocytes differ in sensitivity to a number of environmental toxins and anticancer agents. B lymphocytes are susceptible and T lymphocytes resistant to killing by cyclophosphamide (Cy) metabolites capable of forming DNA interstrand cross-links. However, the mechanisms responsible for the rapid killing and loss of bursal-resident B lymphocytes are unknown. Therefore, we investigated the cellular mechanisms of selective toxicity of two cross-linking drugs, mitomycin C (MMC) and Cy, towards differentiating B and T lymphocyte populations using the chicken embryo model system. Viability of bursal-resident B lymphocytes (bursacytes) decreased starting at 5 h post exposure (PE) to MMC, and was maximally reduced by 71.6% by 10 h PE at the highest dose examined (9.0 micrograms MMC/g). Dose-dependent increases in the percentage of apoptotic bursacytes were observed as early as 5 h PE, and increased to 72% by 10 h PE. This was accompanied by reductions in bursacyte numbers. Cy also induced apoptosis in bursacytes. In contrast, thymus-resident lymphocytes (thymocytes) were much more resistant to the toxic effects of MMC and Cy. Viability of thymocytes was reduced by only 10% in the 9.0 micrograms/g MMC treatment group. In addition, the percentage of thymocytes engaged in apoptosis was much lower than that for bursacytes. MMC induced modest cell cycle inhibition in bursacytes and thymocytes. These data strongly suggest that MMC and Cy-induced diferential toxicity involves primarily early and extensive triggering of apoptosis in differentiating B lymphocytes, leading to rapid reduction of lymphocyte numbers in the embryonic bursa.

Assignment of Rfp-Y to the chicken major histocompatibility complex/NOR microchromosome and evidence for high-frequency recombination associated with the nucleolar organizer region.

Rfp-Y is a second region in the genome of the chicken containing major histocompatibility complex (MHC) class I and II genes. Haplotypes of Rfp-Y assort independently from haplotypes of the B system, a region known to function as a MHC and to be located on chromosome 16 (a microchromosome) with the single nucleolar organizer region (NOR) in the chicken genome. Linkage mapping with reference populations failed to reveal the location of Rfp-Y, leaving Rfp-Y unlinked in a map containing >400 markers. A possible location of Rfp-Y became apparent in studies of chickens trisomic for chromosome 16 when it was noted that the intensity of restriction fragments associated with Rfp-Y increased with increasing copy number of chromosome 16. Further evidence that Rfp-Y might be located on chromosome 16 was obtained when individuals trisomic for chromosome 16 were found to transmit three Rfp-Y haplotypes. Finally, mapping of cosmid cluster III of the molecular map of chicken MHC genes (containing a MHC class II gene and two rRNA genes) to Rfp-Y validated the assignment of Rfp-Y to the MHC/NOR microchromosome. A genetic map can now be drawn for a portion of chicken chromosome 16 with Rfp-Y, encompassing two MHC class I and three MHC class II genes, separated from the B system by a region containing the NOR and exhibiting highly frequent recombination.

Antibody response to sheep red blood cells in major histocompatibility (B) complex aneuploid line of chickens.

An integral part of the immune response is the production of antibodies specific for different antigenic challenges. Genes of the MHC encode products that regulate immunity. This study utilized the FCT-15 line of chickens, which is aneuploid for the chromosome containing the ribosomal RNA genes (rDNA) and the MHC or B complex to determine whether an antibody response to SRBC would vary as a function of B complex gene dose. Mating of trisomic parents (B15B15B15) animals produced progeny having either a disomic (B15B15), trisomic (B15B15B15), or tetrasomic (B15B15B15B15) B complex dosage. The number of B/rDNA chromosomes, and thus the B complex dosage was determined by feather pulp nucleolar typing of chicks at hatch. A 5% SRBC antigenic challenge, which induces a T cell-dependent antibody response, was injected at 6 wk of age. Samples taken prior to SRBC injection as well as 5, 8, and 12 d postinjection were assayed for total and mercaptoethanol-resistant antibody. Peak antibody titers (log2), day of peak titer and rate of titer decline were calculated using a quadratic equation for each bird. Differences among the three B complex dosages were evaluated by analysis of variance. Antibody titers rose from 5 to 8 d postinjection and declined thereafter without significant differences among the three B complex doses. Calculations from the quadratic equations showed that B complex dose affected neither peak antibody titer nor day of peak titer. However, trisomic and tetrasomic animals had significantly more rapid rates of decline from the maximum titer. In aneuploid chickens, changes in antigen processing, antigen presentation, or persistence of processed antigen may maintain levels of antibody production found in disomic chickens and explain the more rapid decline of titer.

Selective expression of major histocompatibility complex (MHC) antigens and modulation of T-cell differentiation in chickens with increased MHC-chromosome dosages.

Increased dosage of genes belonging to the immunoglobulin superfamily may be responsible for some of the less noticeable but targeted phenotypic disturbances seen in trisomy conditions of humans and animals. We used an avian aneuploidy model to study the specific effects of extra major histocompatibility complex (MHC)-microchromosome dosage on the progression of thymocyte differentiation through a broad period of embryonic and neonatal development. The particular goal in the present investigation was to determine whether a reduction in the number of thymocytes, previously observed in the developing thymus of MHC aneuploids, is accompanied by particular alterations in thymocyte differentiation. We hypothesized that the subpopulation structure and/or developmental pattern for thymocyte differentiation are characteristically perturbed (delayed or modified) by increased MHC-chromosome dosage in cells. The regulation of MHC surface antigen expression in aneuploid thymocytes was also studied to detect dosage-dependent expression for one and possibly more sub-regions (class I, II, IV) of the avian MHC. Surface densities of MHC class I antigens on thymocytes were increased significantly at all ages studied, for example by 15% and 45% in trisomics and tetrasomics, respectively at 22 days post-hatching. The surface density of CT1 antigen, a thymocyte-specific marker, was also increased in a dosage-dependent manner, but only in juveniles. Increases in the proportion of alpha beta 1, TCR+ and CD3+ thymocytes were observed in juveniles, with no alterations in other TCR-expressing thymocytes. No major alterations in CD4 and CD8 thymocyte populations were observed. These results demonstrate a targeted effect of extra MHC-chromosome dosage towards enhanced class I and CT1, and not class II or IV, expression. The increased MHC-microchromosome dosage appears to influence primarily immature thymocytes expressing alpha beta 1 TCR and CD3.

Identification of a novel Ca(2+)-regulated protein that is associated with the marginal band and centrosomes of chicken erythrocytes.

We have identified a novel Ca(2+)-regulated protein, p23, that is expressed specifically in avian erythrocyte and thrombocyte lineages. Sequence analysis of this 23 kDa protein reveals that it bears no homology to any known sequence. In mature definitive erythrocytes p23 exists in equilibrium between a soluble and a cytoskeletal bound pool. The cytoskeletal fraction is associated with the marginal band of microtubules, centrosomes and nuclear membrane under conditions of low free [Ca2+]. An increase in free [Ca2+] to 10(-6) M is sufficient to induce dissociation of > 95% of bound p23 from its target cytoskeletal binding sites, yet this [Ca2+] has little effect on calmodulin-mediated MB depolymerization. Analysis of p23 expression and localization during erythropoiesis together with results from heterologous p23 expression in tissue cultured cells demonstrated that this protein does not behave as a bone fide microtubule-associated protein. In addition, the developmental analysis revealed that although p23 is expressed early in definitive erythropoeisis, its association with the MB, centrosome and nuclear membrane occurs only in the final stages of differentiation. This cytoskeletal association correlates with marked p23 stabilization and accumulation at a time p23 expression is being markedly downregulated. We hypothesize that the mechanism of p23 association to the MB and centrosomes may be induced in part by a decrease in intracellular [Ca2+] during the terminal stages of definitive erythropoiesis.

Novel centrosomal protein reveals the presence of multiple centrosomes in turkey (Meleagris gallopavo) bnbn binucleated erythrocytes.

The phenotype of the bnbn hemolytic anemia mutation in the domestic turkey is manifested as binucleation specifically in the definitive erythrocyte lineage, most likely as the consequence of anomolous centrosomal activity (Bloom et al., 1970; Searle and Bloom, 1979). Here we have identified in turkey two variants of the novel, centrosomally-associated erythroid-specific protein p23. One variant is Ca(2+)-sensitive and is highly homologous to its chick counterpart (Zhu et al., 1995, accompanying paper). The other, p21 is a truncated form resulting from a 62 amino acid deletion from the 3' end and a 40 amino acid insertion at the 5' end, and appears to lack Ca(2+)-sensitivity. These proteins are localized at the marginal band, centrosomes and nuclear membrane of differentiated erythrocytes. Anti-p23/p21 immunofluorescence revealed the presence of multiple centrosomes in bnbn erythrocytes. We therefore undertook a detailed genetic analysis to determine whether the p21 variant represented the bn mutation. Initial tests of normal BnBn and mutant bnbn individuals suggested that the p23/p21 proteins might be encoded by the Bn/bn genes. However, further genetic tests demonstrated independent segregation for these two genetic loci. Thus, these proteins are encoded by the heretofore undescribed genes, p23/p21, mapping to an autosomal locus in the turkey genome.

Teratogenic development in chicken embryos associated with a major deletion in the rRNA gene cluster.

A new strain of chickens (mPNU) that segregates a severely deleted rDNA cluster was studied. Individuals heterozygous (+/p2 ) and homozygous (p2 /p2 ) for the deletion were found to have 56 and 27%, respectively, of the normal complement of rRNA genes (290 copies/cell). Morphogenesis, cellular rRNA levels, and nucleolar sizes, were investigated and compared in normal +/+, +/p2 , and p2 /p2 embryos. Cellular rRNA contents were similar among the three genotypes at stage X, but subsequently during gastrulation, p2 /p2 levels were reduced to 56% and eventually to 43% of +/+. Viability and morphogenesis were normal in p2 /p2 embryos until the initial primitive streak stage of gastrulation. However, further development was abnormal and characterized by disrupted axis formation. In +/+ and +/p2 embryos, rRNA levels and nucleolar sizes increased during early development; however, the profile of these increases differed temporally and quantitatively between the genotypes. The +/p2 embryos, at the full streak stage of gastrulation, exhibited reduced rRNA levels and nucleolar sizes (80% of +/+), yet the +/p2 embryos developed normally. These studies establish a minimum copy number requirement lower than previously demonstrated, that is, a rDNA genotype with only 56% of the normal gene complement (∼160 genes) is compatible with early embryonic viability. Also, a rRNA threshold was detected: rRNA levels that were 56% of +/+ failed to support normal gastrulation; however, even under the circumstance of reduced rRNA levels (43% of control), some aspects of gastrulation apparently continue (cell migration and invagination). The teratogenic development of p2 /p2 embryos is a biological consequence unique from that found in other metazoan models of rDNA-deficiency, and will be useful as a model to investigate mechanisms of vertebrate gastrulation and axis formation.

Detection and localization of 3,3',4,4'-tetrachlorobiphenyl-induced P4501A protein in avian primary immune tissues.

P4501A can be detected in thymic and bursal microsomes from chickens pretreated with 3,3',4,4'-tetrachlorobiphenyl (TCB) using a polyclonal antibody against purified P4501A from 3-methylcholanthrene (3-MC)-induced chicken embryo liver. A dose-response for induction by TCB of P4501A protein was detected by Western blotting in both bursal and thymic microsomes. Ethoxyresorufin-O-deethylase (EROD), a specific catalytic activity of P4501A, was also induced in a dose-response fashion. More TCB-induced P4501A was detected in thymus than bursa by both methods. No EROD was detected in bursal or thymic microsomes from untreated chickens, although P4501A protein was detected at very low levels in thymic microsomes from untreated chickens. P4501A was detected by immunohistochemistry in scattered patches of non-lymphocytic cells residing in medullary regions of the TCB-induced thymus but was not detected in lymphocytes. This result supports previous work demonstrating that TCB-inducible EROD is much higher in the supporting tissue cell fractions than in lymphocyte fractions of the primary immune tissues. Although EROD was induced by TCB in the late stage embryo after 20 h exposure, no effect of TCB on the cell cycle in thymic or bursal lymphocytes was observed over the same period. The same TCB exposure resulted in bursal but not thymic cellular depletion. Thymic and bursal supporting tissue cells may be primary sites of immunosuppression within these organs by P4501A inducers or substrates whether immunosuppression occurs subsequent to metabolism or through interaction with Ah receptors.

Cytogenetic studies of cell lines derived from Marek's disease virus-induced local lesions.

Cytogenetic analysis of cell lines derived from Marek's disease virus (MDV) induced local lesions was performed to detect the presence of a chromosomal alteration found previously in cell lines derived from MDV-induced visceral tumors. This chromosomal alteration involves an amplified region on the short arm of chromosome 1. Trypsin G-binding was performed on 12 local-lesion cell lines having various T-cell receptor phenotypes. Eight of 12 cell lines had a diploid female karyotype, and four lines were diploid males. Ten of the cell lines showed a normal G-banding pattern; two lines, however, had the short arm amplification on chromosome 1 (1p+). Thus, the 1p+ alteration does not appear to be necessary for establishment of cell lines from local lesions. The occurrence of the 1p+ alteration in Marek's disease cell lines suggests a possible role for this alteration in late stages of multi-step viral oncogenesis.

Nucleolar size polymorphisms in commercial layer chickens; determination of incidence, inheritance, and nucleolar sizes.

In the chicken, one chromosome pair encodes the ribosomal (r)RNA genes and two nucleoli are formed in interphase cells. The nucleolus is the site of rRNA synthesis and ribosome biogenesis. Nucleolar size polymorphisms have been detected in research strains of chickens and found to represent heterozygosity for rRNA gene copy number. The objectives of the present study were to determine whether nucleolar size polymorphisms exist in commercial chicken flocks and whether such polymorphisms are under genetic control. The occurrence of nucleolar size polymorphisms was studied in three lines of commercial layer chickens (designated A, B, and C). Nucleolar size polymorphisms were found in all three lines. However, the lines differed in the proportion of individuals exhibiting the polymorphic phenotype of two unequal-sized nucleoli (2P). The 2P phenotype, determined in successive years, was found in 10 to 14% of Line A birds, 18 to 23% of Line B birds, and 41 to 63% of Line C birds. The inheritance pattern of the nucleolar size polymorphism and nucleoli sizes were studied in Line C birds. The 2P phenotype was found to be inherited in Mendelian fashion. The large nucleolus was 1.7x and 1.6x that of the smaller nucleolus in 2P males and females, respectively, and was larger than the nucleoli of 2E birds (2E = nonpolymorphic phenotype of two equal-sized nucleoli). Total nucleolar size was greater in cells of 2P birds than in cells of 2E birds. These data suggest that the genetic basis for the large nucleolus in Line C 2P cells is a "larger than normal" rDNA cluster.

Effects of rRNA gene copy number and nucleolar variation on early development: inhibition of gastrulation in rDNA-deficient chick embryos.

Because of their structural and catalytic functions during protein synthesis, the 18S, 5.8S, and 28S ribosomal RNAs (rRNAs) are essential for the support of differentiation, development, and growth. The genes encoding these RNAs are present in high copy number in all eukaryotes. Although there is evidence for the existence of variation for rRNA gene copy number within higher vertebrate species, there is little knowledge concerning the effects of such variation, especially reductions, on development and viability of homeothermic vertebrates. The main objective of this study was to determine the developmental potential of chick embryos containing defined deficiencies for rRNA gene copy number in order to assess the contribution of rDNA cluster size variation to embryonic mortality in homeothermic vertebrates. This was achieved by studying a strain of chickens containing nucleolar size polymorphisms that reflect rDNA cluster size polymorphisms. Embryos exhibiting a nucleolar phenotype of one large and one very small nucleolus (Pp) are heterozygous for a reduced rDNA cluster (+/p1) and were shown in the present study to contain about 66% of the complement of rRNA genes in normal individuals (+/+) that show two large equal-sized nucleoli (PP). The +/p1 embryos were found to develop and grow normally. Embryos exhibiting a nucleolar phenotype of two very small nucleoli (pp) are homozygous for the rDNA-deficient cluster (p1/p1) and contained about 45% of the normal rDNA complement of genes. These p1/p1 embryos were arrested in their development during early gastrulation. They exhibited a characteristic morphology consisting of a dorsal invagination that was strikingly different from the primitive streak formed in +/+ and +/p1 individuals.(ABSTRACT TRUNCATED AT 250 WORDS)

Consistent chromosomal aberration in cell lines transformed with Marek's disease herpesvirus: evidence of genomic DNA amplification.

A specific chromosomal aberration was observed in 14 of 15 avian lymphoblastoid cell lines transformed with Marek's disease herpesvirus. This aberration, designated dup(1p)(p22-p23), appeared as an extra G-positive band and interband on the short arm of one chromosome I homolog. Using fluorescent in situ hybridization, we identified amplified genomic DNA sequences in this region. This amplification involves sequences linked to an endogenous retrovirus locus and genes in the histone multigene family. This aberration was not observed in cells transformed by reticuloendotheliosis virus or by avian leukosis virus, nor has it been observed in untransformed chicken cells. The induction of the 1p+ chromosomal aberration may be an essential event in the transformation of lymphocytes by Marek's disease virus.

Islet amyloid polypeptide-like immunoreactivity in human tissue and endocrine tumors.

We have detected islet amyloid polypeptide (IAPP)-like immunoreactivity (-LI) in human pancreas and in a range of endocrine tumors, including oat cell carcinoma of the lung and pancreatic tumours producing insulin, gastrin, glucagon, and vasoactive intestinal peptide. Gel permeation chromatography of the IAPP-LI revealed that, except in the carcinoid, more than 80% coeluted with synthetic human IAPP. The remaining immunoreactivity consisted of variable amounts of larger and smaller molecular forms. The concentration of IAPP-LI in the circulation of patients with diagnosed pancreatic endocrine tumors was not significantly elevated above normal fasting levels. IAPP is, therefore, produced by a range of endocrine tumors and may relate to the deposition of endocrine amyloid.

Selective aflatoxin B1-induced sister chromatid exchanges and cytotoxicity in differentiating B and T lymphocytes in vivo.

The purpose of this study was to assess the genotoxic and cytotoxic effects of the fungal metabolite aflatoxin B1 (AfB1) on the developing immune system of the chick embryo, a model in vivo system. Of particular interest was the assessment of AfB1-mediated selective toxicity toward developing B lymphocytes as compared to T lymphocytes. In vivo bromodeoxyuridine (BrdU) labelling of DNA was used to detect the induction of sister chromatid exchanges (SCE) in lymphocytes and to assess the progression of these cells through successive cell cycles. Cytotoxicity was also assessed by studying the entrance and maintenance of cells in mitosis (mitotic index). Graded doses of AfB1 (1.09-17.4 micrograms/g embryo) were applied to chick embryos at 18 days of incubation (DI). Embryos also received two doses of BrdU at 3 mg/200 microliters (3 hr apart) to provide continuous labelling of B and T lymphocyte replicating DNA. B and T lymphocytes were harvested 20 hr post-AfB1/BrdU exposure from the bursa and thymus, respectively, and were processed for cytogenetic analyses. AfB1 induced dose-related increases in SCE in B lymphocytes; this induction was 6- to 8-fold that of controls at the higher doses tested. AfB1-mediated induction of SCE in T cells was just 2-fold that of controls at the highest dose tested. AfB1 reduced the progression of B cells and to a lesser extent T cells through successive rounds of replication. Furthermore, AfB1 dramatically reduced the mitotic index of B cells but not of T cells. These data indicate both selective genotoxicity and cytotoxicity of AfB1 toward B cells in the late stage embryo.