Induction of micronuclei by HTLV-I Tax: a cellular assay for function

Cellular chromosomal damage is ubiquitously seen in HTLV-I-transformed lymphocytes. It is also characteristic of cells that have been exposed to mutagens. A sensitive measurement for mutagen-induced DNA damage is the formation of micronuclei in treated cells. Because current evidence suggests that HTLV-I Tax is etiologically linked to transformation, we tested for its activity in inducing micronuclei. We show here that transfection into cells of a Tax-producing plasmid rapidly induced the formation of micronuclei. This effect cooperated with that of a mutagen (mitomycin C) and was correlated with the inherent trans-activation capacity of Tax. These findings suggest that a commonly used mutagen assay could be a quick biological test for putatively oncogenic proteins.     

Cell-type-specific transactivation of the parathyroid hormone-related protein gene promoter by the human T-cell leukemia virus type I (HTLV-I) tax and HTLV-II tax proteins

The human T-cell leukemia virus type I (HTLV-I) and HTLV-II Tax proteins are potent transactivators of viral and cellular gene expression. Using deletion mutants, the downstream parathyroid hormone-related protein (PTHrP) promoter is shown to be responsive to both HTLV-I and HTLV-II Tax as well as the AP1/c-jun proto-oncogene. Transactivation of PTHrP by Tax was seen in T cells but not in B-cell lines or fibroblasts. A carboxy terminal Tax deletion mutant was deficient in transactivation of both the PTHrP and IL2R alpha promoters but not the HTLV-I long terminal repeat (LTR). Exogenous provision of NFkB rescued IL2R alpha expression but not the PTHrP promoter. Thus, HTLV-I Tax, HTLV-II Tax, and c-jun transactivate PTHrP and may contribute to the pathogenesis of hypercalcemia in adult T-cell leukemia.     

HTLV-I Tax protein binds to MEKK1 to stimulate IkappaB kinase activity and NF-kappaB activation

NF-kappaB, a key regulator of the cellular inflammatory and immune response, is activated by the HTLV-I transforming and transactivating protein Tax. We show that Tax binds to the amino terminus of the protein kinase MEKK1, a component of an IkappaB kinase complex, and stimulates MEKK1 kinase activity. Tax expression increases the activity of IkappaB kinase beta (IKKbeta) to enhance phosphorylation of serine residues in IkappaB alpha that lead to its degradation. Dominant negative mutants of both IKKbeta and MEKK1 prevent Tax activation of the NF-kappaB pathway. Furthermore, recombinant MEKK1 stimulates IKKbeta phosphorylation of IkappaB alpha. Thus, Tax-mediated increases in NF-kappaB nuclear translocation result from direct interactions of Tax and MEKK1 leading to enhanced IKKbeta phosphorylation of IkappaB alpha.     

A transgenic mouse model for mammary carcinogenesis

Missense mutations in the p53 tumor suppressor occur frequently in human breast cancer and influence both the prognosis and response to chemotherapy. Amino acid 175 (equivalent to murine 172) is the second most common site of missense mutations in p53 in human breast cancer. Over 95% of these mutations are arginine-to-histidine (R-H) substitutions resulting in a gain-of-function, and not merely a dominant-negative phenotype. Transgenic mice expressing a p53 172(R-H) construct targeted to the mammary gland by means of a whey acidic protein (WAP) promoter were characterized as a model system in order to determine the specific effects of this mutation on mammary tumorigenesis. Although transgene expression alone had no apparent effect on normal mammary development, transgenic mice treated with the chemical carcinogen dimethylbenz(a)anthracene developed tumors with much shorter latency than did control littermates and had a greater tumor burden. Tumors arising in transgenic mice did not exhibit either decreased apoptosis or increased cell proliferation relative to tumors arising in nontransgenic littermates, but did display increased genomic instability. Large pleiomorphic nuclei were visible in many tumors from transgenic mice, and DNA flow analysis confirmed the presence of significant aneuploid cell populations. Since these transgenic mice develop very few spontaneous tumors, while accelerating carcinogen-and oncogene-mediated tumorigenesis, this mouse model will, therefore, be useful in the investigation of early events in mammary tumorigenesis. It may also be used as a preclinical model to test newly developed chemotherapeutic strategies.     

Tat-induced lesions in transgenic mice do not correlate with the HIV-1 LTR transactivation

A pilot study was conducted in 7 normal volunteers to demonstrate the feasibility of employing pharmacokinetic tailoring to achieve matching plasma opioid concentration-time curves after epidural (e.p.) and intravenous (i.v.) alfentanil administration. Each subject participated in 1 pretest and 2 test sessions. Our pain model was cutaneous electrical stimulation of the finger and toe, adjusted to produce a baseline pain report of 5 (strong pain on a 0-5 scale). On test day 1, subjects received e.p. alfentanil (750 micrograms) and an i.v. saline infusion. Serial measurements of analgesia, end tidal CO2, pupil size, subjective side effects, and plasma alfentanil concentrations were conducted before and at various time intervals over a 4-h period after alfentanil administration. On test day 2, subjects received e.p. saline and a pharmacokinetically tailored i.v. infusion (using individual pharmacokinetics determined on the pretest day) designed to achieve a plasma concentration-time profile identical to that observed on the epidural day. The same battery of effect measurements was administered as on the 1st test day. Plasma alfentanil was measured to verify the accuracy of the tailored infusion. Plasma alfentanil concentration profiles were nearly identical on both test days. Peak plasma alfentanil concentrations were near the reported minimum effective analgesic concentration (MEAC). Overall, analgesia was slightly greater with e.p. administration. Onset of pain relief was rapid, and duration was approximately 1.5 h with e.p. and 1 h with i.v. alfentanil. There were no differences in pupil size, ETCO2, or subjective side effects between e.p. versus i.v. administration. We conclude that systemic redistribution from the epidural space appears to account for most, but not all, of the analgesia.     

A transgenic model to analyze the immunoregulatory role of IL-10 secreted by antigen-presenting cells

IL-10 is a cytokine secreted by a wide variety of cells type that has pleiotropic stimulatory and suppressive activities on both lymphoid and myeloid cells in vitro. To analyze the consequences of high IL-10 secretion by APCs in immune responses, we produced transgenic mice expressing human IL-10 directed by the MHC class II Ea promoter. Despite alterations in the development of T and B cells, no gross abnormalities were detected in peripheral lymphocyte populations or serum Ig levels. However, when immunized using conditions that give either a Th2-type or a Th1-type response, IL-10 transgenic mice failed to mount a significant T or B cell immune response to OVA. IL-10 transgenic mice were also highly susceptible to infection with intracellular pathogens like Listeria monocytogenes or Leishmania major, in contrast to IL-10 transgenic mice, where the transgene was express in T cells. Finally, the recently described stimulatory effect of IL-10 on CD8+ T cells was confirmed by the ability of IL-10 transgenic mice to limit the growth of immunogenic tumors by a CTL-mediated mechanism. These results demonstrate, that, depending on the type of immune response, IL-10 can mediate immunosuppressive or immunostimulatory activities in vivo.     

Suppression of skin reactivity to purified protein derivative by hepatitis C virus among HTLV-I carriers in Japan

Gamma-aminobutyric acid (GABA)A receptors are the sites of action for many antiepileptic drugs such as benzodiazepines and barbiturates. We report the results of molecular cloning of the gamma1-subunit from seizure prone DBA/2J and resistant C57BL/6J inbred mice, and analyses of nucleotide sequences and expression of the gamma1-subunit messenger RNA (mRNA) in DBA/2 and C57BL/6 inbred mice. The mouse gamma1-subunit complementary DNA (cDNA) shares 98% similarity with that of the rat at the level of amino acid sequence. Northern blot hybridization indicates that the gamma1-subunit mRNA is expressed predominantly in areas other than the cerebral cortex and cerebellum and shows little change with postnatal development. No differences have been found for the subunit between DBA/2 and C57BL/6 mice either for nucleotide sequence or for level of expression of the subunit's mRNA in whole brain by Northern blots at 3 weeks of age.     

A transgenic mouse model of metastatic prostate cancer originating from neuroendocrine cells

A transgenic mouse model of metastatic prostate cancer has been developed that is 100% penetrant in multiple pedigrees. Nucleotides -6500 to +34 of the mouse cryptdin-2 gene were used to direct expression of simian virus 40 T antigen to a subset of neuroendocrine cells in all lobes of the FVB/N mouse prostate. Transgene expression is initiated between 7 and 8 weeks of age and leads to development of prostatic intraepithelial neoplasia within a week. Prostatic intraepithelial neoplasia progresses rapidly to local invasion. Metastases to lymph nodes, liver, lung, and bone are common by 6 months. Tumorigenesis is not dependent on androgens. This model indicates that the neuroendocrine cell lineage of the prostate is exquisitely sensitive to transformation and provides insights about the significance of neuroendocrine differentiation in human prostate cancer.     

Differential transactivation by the androgen receptor in prostate cancer cells

BACKGROUND: The purpose of this study was to determine the contribution of different transactivating regions of the androgen receptor (AR) to the induction of androgen-regulated promoters in poorly (PC3 cells) and well-differentiated (LNCaP cells) prostate cancer cell lines. METHODS: PC3 and LNCaP cells were co-transfected with plasmids expressing full-length AR or deletion mutants together with luciferase reporters linked to the probasin (PB) and PSA promoters; as well as to ARR3tk, a PB-derived recombinant promoter. RESULTS: Androgen induction of the ARR3tk promoter in the presence of AR was 8- to 10-fold higher than that seen with the PB promoter. Activation of ARR3tk was greatest with an androgen-independent construct in which the first 231 amino acids and the ligand binding domain had been removed, indicating that this promoter is more responsive to activating functions in the N-terminal domain than in the ligand binding domain. By comparison, induction of the PB promoter was greatest with the full-length AR, which suggests that the ligand binding domain also makes a major contribution to the activation of this promoter. In similar analyses with the PSA promoter, AR regions required for promoter induction was dependent on the host cell type. In PC3 cells, the predominant AR transactivation function was androgen-independent and resided in the N-terminal domain, whereas in LNCaP cells, the highest level of induction was androgen dependent and also required participation of the ligand binding domain. CONCLUSIONS: Our results indicate that the relative utilization of transactivating functions in N-terminal and ligand binding domains of the AR is promoter and cell specific.     

Involution of the lactating mammary gland is inhibited by the IGF system in a transgenic mouse model

Development of the mammary gland during puberty, pregnancy, and lactation is controlled by steroid and peptide hormones and growth factors. To determine the role of the insulin-like growth factors (IGFs) in this process we developed a transgenic model using the whey acidic protein (WAP) gene to direct expression of rat IGF-I and human IGF binding protein-3 (IGFBP-3) to mammary tissue during late pregnancy and throughout lactation. High levels of expression of transgenic IGF-I and IGFBP-3 were seen in lobular-alveolar cells by in situ hybridization. There was no obvious effect on mammary development during pregnancy and lactation; indeed, mothers were capable of nursing their pups normally and the only structural difference seen in the mammary glands at peak lactation was an overall smaller size of the alveoli. We also evaluated the role of IGF-I and IGFBP-3 in the remodeling of mammary tissue during involution. Compared with control animals, the process of involution was modified in both transgenic lines. The degree of apoptotic cells was lower in the WAP-IGF-I and WAP-BP-3 expressing mice. In addition, there was a more quiescent pattern of involution with residual lobular secretary ability and a muted host inflammatory reaction with fewer lumenal microcalcifications. These results demonstrate that IGF-I and IGFBP-3 may modulate the involutionary process of the lactating mammary gland.     

Adenovirus E1A expression controlled by the red pigment promoter in transgenic mice: a model for developmental abnormalities of the eye

Four transgenic founder mice were produced that carry the adenovirus E1A gene under control of the human red pigment promoter. Two of the founder animals passed the transgene to progeny, and one line was bred to homozygosity. The line of mice homozygous for the transgene expressed E1A-specific RNA at a low level in their eyes; no expression could be detected in other tissues that were tested. Either the founder animals or their progeny exhibited developmental abnormalities of their eyes, including a small eye phenotype, retinal dysplasia, anterior cleavage syndrome, and retinal pigment epithelium dysplasia. Individuals from the same line of mice exhibited subsets of the abnormalities, e.g., many animals had one normal size eye and one small eye, even though both eyes contained E1A-specific RNA. No tumors resulted from E1A expression in animals monitored for 22 months.