Molecular regulation of genes encoding xenobiotic-metabolizing enzymes: mechanisms involving endogenous factors.

It is widely recognized that xenobiotic-metabolizing enzymes play a fundamental role in the basic processes of carcinogenesis and toxicity on one hand, and chemoprevention and drug efficacy on the other. Realization that different factors can profoundly affect the expression of these enzymes at the genome level has resulted in an enhanced appreciation of the importance these genes play in our modern industrialized age. There continues to be rapid proliferation of studies addressing the molecular regulation of these genes. The discovery of common signal transduction pathways and transcription factors that dictate tissue and developmental-specific expression, as well as variation in expression within a given tissue, suggest that there may be significant interaction among these various regulatory systems. This report is a summary of a symposium that was part of the Structure, Function and Regulation of Cytochromes P450 and Xenobiotic Metabolizing Enzymes satellite meeting of the 2000 joint meeting of the American Society for Biochemistry and Molecular Biology, the American Society for Pharmacology and Experimental Therapeutics, the French Pharmacological Society, and the Pharmacological Society of Canada held in Boston, Massachusetts. This symposium brought together several speakers who addressed specific receptor-mediated signal transduction pathways involved in the regulation of xenobiotic-metabolizing enzymes, as well as other molecular mechanisms whereby endogenous factors are involved in controlling tissue- and developmental-specific expression.

Maximal aryl hydrocarbon receptor activity depends on an interaction with the retinoblastoma protein.

The aryl hydrocarbon receptor (AhR) belongs to the basic helix-loop-helix/periodicity/AhR nuclear translocator/simple-minded (Per-Arnt-Sim) family of transcription factors that regulate critical functions during development and tissue homeostasis. Within this family, the AhR is the only member conditionally activated in response to ligand binding, typified by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). We recently demonstrated that the AhR interacts with the retinoblastoma protein (pRb). This report presents evidence that a LXCXE motif in the AhR protein confers pRb binding, which is necessary for maximal TCDD induced G(1) arrest in rat 5L hepatoma cells. The data support a mechanism whereby pRb seems to regulate G(1) cell cycle progression distinct from the direct repression of E2F-mediated transcription. Furthermore, the results indicate that the AhR-pRb interaction regulates TCDD induction of CYP1A1, suggesting that pRb may be a general AhR coactivator.

Insulin-like growth factor I is a dual effector of multiple myeloma cell growth.

Multiple myeloma (MM) is an invariably fatal disease that accounts for approximately 1% to 2% of all human cancers. Surprisingly little is known about the cellular pathways contributing to growth of these tumors. Although the cytokine interleukin-6 has been suggested to be the major stimulus for myeloma cell growth, the role of a second potential growth factor, insulin-like growth factor I (IGF-I), has been less clearly defined. The IGF-I signaling cascade in 8 MM cell lines was examined. In 7 of these, the IGF-I receptor (IGF-IR) was expressed and autophosphorylated in response to ligand. Downstream of IGF-IR, insulin receptor substrate 1 was phosphorylated, leading to the activation of phosphatidylinositol-3'-kinase (PI-3K). PI-3K, in turn, regulated 2 distinct pathways. The first included Akt and Bad, leading to an inhibition of apoptosis; the second included the mitogen-activated protein kinase (MAPK), resulting in proliferation. Biologic relevance of this pathway was demonstrated because in vitro IGF-I induced both an antiapoptotic and a proliferative effect. Importantly, in vivo administration of IGF-I in SCID mice inoculated with the OPM-2 line led to approximately twice the growth rate of tumor cells as in controls. These results suggest that IGF-I activates at least 2 pathways effecting myeloma cell growth and contributes significantly to expansion of these cells in vivo. (Blood. 2000;96:2856-2861)

Expression of PTEN in PTEN-deficient multiple myeloma cells abolishes tumor growth in vivo.

Biochemical abnormalities associated with the development of multiple myeloma have been difficult to define especially in terms of demonstrating an in vivo effect of suspected lesions. Herein, we have identified such a defect associated with lack of expression of PTEN, a cellular phosphatase involved in the regulation of phosphatidylinositol phosphates (PIP's). In myeloma cells, PIP's are required for phosphorylation of Akt, a key event leading to inhibition of apoptosis. Loss of PTEN results in a failure to de-phosphorylate PIP's and a corresponding increase in Akt phosphorylation. OPM-2 cells lacking PTEN expression have the highest level of Akt phosphorylation of eight lines examined. Loss of PTEN was found to be associated with a 630 bp deletion corresponding to amino acids 56 - 267. Ectopic expression of wild type PTEN in OPM-2 cells inhibited Akt phosphorylation which was correlated with an increase in apoptosis. The in vivo relevance of loss of PTEN expression was demonstrated by injecting control and wild type PTEN transfected OPM-2 cells into SCID mice. Tumors arose at an incidence of 100% in controls, but only 50% (and of smaller size and longer latency) in low PTEN expressing clones. Importantly, clones expressing high levels of PTEN failed to produce tumors even at five times the latency period of controls. These results demonstrate that PTEN deletion/mutation is responsible for in vivo growth of this tumor and suggests that PTEN regulation may play an important role in tumor development in a subset of multiple myeloma patients. Oncogene (2000) 19, 4091 - 4095

A direct interaction between the aryl hydrocarbon receptor and retinoblastoma protein. Linking dioxin signaling to the cell cycle.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor in eukaryotic cells that alters gene expression in response to the environmental contaminant 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD). In 5L hepatoma cells, TCDD induces a G1 cell cycle arrest through a mechanism that involves the AhR. The retinoblastoma tumor suppressor protein (pRb) controls cell cycle progression through G1 in addition to promoting differentiation. We examined whether the human AhR or its dimerization partner, the AhR nuclear translocator, interacts with pRb as a basis of the TCDD-induced cell cycle arrest. In vivo and in vitro assays reveal a direct interaction between pRb and the AhR but not the AhR nuclear translocator protein. Binding between the AhR and pRb occurs through two distinct regions in the AhR. A high affinity site lies within the N-terminal 364 amino acids of the AhR, whereas a lower affinity binding region colocalizes with the glutamine-rich transactivation domain of the receptor. AhR ligand binding is not required for the pRb interaction per se, although immunoprecipitation experiments in 5L cells reveal that pRb associates preferentially with the liganded AhR, consistent with a requirement for ligand-induced nuclear translocation. These observations provide a mechanistic insight into AhR-mediated cell cycle arrest and a new perspective on TCDD-induced toxicity.

Development of a non-radioactive DNA probe and in situ hybridization for detection of Anaplasma marginale in ticks and cattle.

A non-radioactive DNA probe was developed for detection of Anaplasma marginale in ticks and cattle. The probe was labeled with digoxigenin 11-dUTP by polymerase chain reaction. The probe was tested on bovine blood and was found to be a sensitive and specific detection method for A. marginale in cattle. The DNA probe was then adapted for in situ hybridization (ISH) of A. marginale in Dermacentor andersoni and D. variabilis ticks infected either as nymphs or adults. One-half of each tick was studied with ISH while the other half was examined with light and electron microscopy. In male ticks infected as adults, tick gut cells first became infected with A. marginale while ticks fed on an infected calf, and they remained infected as they transmission fed on a second, susceptible calf. At the onset of transmission feeding, salivary glands became infected with A. marginale. During transmission feeding infection was also observed in interstitial, reproductive, skeletal muscle, fat body and Malpighian tubule tissue, resulting in a generalized A. marginale infection. When adult ticks that acquired infection as nymphs were examined with ISH and microscopy, gut tissues of both D. andersoni and D. variabilis became infected with A. marginale. However, salivary gland infection was seen only in D. variabilis, even though both species of ticks transmitted A. marginale to susceptible calves. A. marginale was not seen with ISH or microscopy in hemocytes collected from both species of ticks and, thus, hemocytes do not appear to play a role in the development of A. marginale in ticks.

Use of a nonradioactive DNA probe for detection of Anaplasma marginale infection in field cattle: comparison with complement fixation serology and microscopic examination.

A sensitive Anaplasma marginale-specific 409-base pair DNA probe was developed in a previous study for detection of A. marginale infection in experimentally infected cattle with a test that employed slot-blot and in situ hybridization. To test the suitability of the probe to detect A. marginale in the blood of naturally infected carrier cattle, slot-blot hybridization was used to determine the infection rate of A. marginale in cattle from 3 geographic areas in Oklahoma. For comparison, blood samples from the same cattle were also examined by light microscopy and were tested by the complement fixation test. For the DNA hybridization assay, the probe was labeled with digoxigenin 11-dUTP by polymerase chain reaction (PCR). DNA was extracted from blood using the QIAamp blood kit and then applied to a nylon membrane and hybridized with the probe. The study herds consisted of 31 beef cows in Harper County, OK, and 42 and 70 dairy cows from Payne and Pittsburg counties, OK, respectively. In the 3 herds, 80.6%, 92.8%, and 57.1% of the cows were positive for A. marginale as assessed with the DNA hybridization assay. In contrast, only 25.8% and 2.86% were complement fixation positive in 2 herds, and no complement fixation positives were found in 1 herd. Uncountable parasitemia that was too low to accurately determine (< 0.01%) from 29.0%, 4.8%, and 11.4% of the samples, respectively, was demonstrated by microscopic examination. All samples positive by complement fixation and microscopic examination had positive probe reactions in the DNA hybridization assay. Therefore, the PCR-mediated nonradioactive DNA probe described here may be useful in epidemiologic investigations and in identification of carrier cattle. This assay could be adapted for use in diagnostic laboratories because it is sensitive, specific, nontoxic, quickly executed, and inexpensive.

Developmental studies of Anaplasma marginale (Rickettsiales:Anaplasmataceae) in male Dermacentor andersoni (Acari:Ixodidae) infected as adults by using nonradioactive in situ hybridization and microscopy.

The development of Anaplasma marginale Theiler was studied in ticks using a nonradioactive in situ hybridization method developed in our laboratory. Male Rocky Mountain wood ticks, Dermacentor andersoni Stiles, were infected intrastadially by allowing them to feed for 7 d on an infected calf (acquisition feeding). The ticks were then removed and held in a humidity chamber for 5 d before being fed on a 2nd susceptible call for 10 d (transmission feeding). Two groups of 10 ticks were collected daily during the 22-d experiment. In one group one-half of each tick was processed and embedded in paraffin and in the other group one-half of each tick was embedded in LR White for in situ hybridization. The companion tick halves from each group were fixed and embedded in Dow Epoxy Resin resin for routine light and electron microscopy. As detected by in situ hybridization on LR White- and paraffin-embedded sections and by microscopy, initial infection of A. marginale in ticks occurred in gut tissues either on the 7th d of acquisition feeding or the 1st d of the held period and infection persisted throughout transmission feeding. The highest number of ticks with gut infection was observed on the 5th d of transmission feeding. Salivary glands became infected with A. marginale on the 1st day of transmission feeding and remained infected throughout the transmission feeding period. Peak infection was observed on day 4 of transmission feeding. After the beginning of transmission feeding, A. marginale infection was also observed in interstitial, reproductive, skeletal muscle, fat body, and Malpighian tubule tissues. Although A. marginale infection of ticks clearly originates in midgut epithelial cells, many tissues eventually become infected during transmission feeding, resulting in a generalized infection. The infection of multiple tissues may contribute to the ability of A. marginale infection to persist in intrastadially infected male ticks.

Establishment of the tick (Acari:Ixodidae)-borne cattle pathogen Anaplasma marginale (Rickettsiales:Anaplasmataceae) in tick cell culture.

Anaplasma marginale is a tick-borne rickettsia that causes bovine anaplasmosis worldwide. Despite its importance, A. marginale has thus far not been established in a continuous culture system. We have propagated A. marginale continuously for the 1st time in a tick cell line derived from the black-legged tick, Ixodes scapularis Say, using infected bovine blood as the inoculum. Erythrocytic stages invaded the tick cells and multiplied in membrane-lined vacuoles to form colonies typical of those observed in naturally infected ticks as demonstrated by light and electron microscopy. The rickettsiae have been passaged serially for 3 yr and have been cryopreserved in liquid nitrogen. Antigens present in A. marginale from tick cell culture were recognized by bovine immune serum against the blood stages of A. marginale. A. marginale grown in this tick cell line was infective for calves, and male ticks fed on the calves transmitted A. marginale to a susceptible calf. The ability to culture A. marginale removes a major impediment to the study of Anaplasma biology in vitro, and will enhance development of vaccines and diagnostic tests.

Detection of Anaplasma marginale DNA in bovine erythrocytes by slot-blot and in situ hybridization with a PCR-mediated digoxigenin-labeled DNA probe.

A 409-base pair (bp) DNA fragment derived from the msp-1 beta gene of Anaplasma marginale was amplified and simultaneously labeled with digoxigenin-11-dUTP by a polymerase chain reaction (PCR) assay. The resulting digoxigenin-labeled 409-bp PCR product was used as a probe for slot-blot and in situ hybridization to detect A. marginale DNA from experimentally infected bovine erythrocytes. The hybrid formation was detected with alkaline phosphatase-conjugated anti-digoxigenin antibody and substrates 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium salt. In slot-blot hybridizations, the probe detected A. marginale DNA from approximately 1,000-10,000 infected erythrocytes in 1.25 ml of whole blood, which is equivalent to a parasitemia level of 0.00001%. The probe proved to be a A. marginale-specific when tested with 17 species of microorganisms. The applicability of the probe for diagnosis was tested by screening A. marginale infections in 2 experimentally infected splenectomized cattle before microscopically detectable parasitemias and after acute infection. After inoculation of infected blood, A. marginale infections were detected with the probe 14 days prior to detection in stained smears. Microscopically inapparent parasitemias were also detected with the probe for 2 months after acute disease. When the probe was used for in situ hybridization on methanol-fixed blood smears, probe reaction could be visualized with light microscopy on A. marginale inclusions within infected erythrocytes. The probe reaction was not observed on leukocytes and uninfected erythrocytes from infected blood smears, on erythrocytes from uninfected blood samples, or on samples infected with A. ovis, Babesia bovis, or B. bigemina. This PCR-mediated nonradioactive probe appears to be a sensitive diagnostic test for A. marginale.

A survey of phlebotomine sandflies (Diptera: Psychodidae) in Hubei Province, China.

From June to August, 1987 a survey of phlebotomine sandflies was carried out by means of aspirator and light-sticky traps in nine counties viz., Yunxi, Wudangshan, Snennongjia, Zigui, Yichang, Lichuan, Laifeng, Hongan and Yangxin, Hubei Province. A total of nine species had been identified: Phlebotomus chinensis, P. kiangsuensis, P. mongolensis, Sergentomyia anhuiensis, S. koloshanensis, S. nankingensis, S. squamirostris, S. zhengjiani and S. barraudi. Their distribution was summarized in a map.