Characterization of the androgen receptor from a Syrian hamster ductus deferens tumor cell line (DDT1).

The hamster ductus deferens cloned tumor cell line (DDT1) contains a complex steroid receptor protein that binds 3H-labeled 5 alpha-dihydrotestosterone,. Diethylaminoethyl (DEAE) chromatography of cytosol from these cells yields two major receptor peaks of activity. Identification of this steroid binding protein as a cytoplasmic receptor was confirmed by salt dissociation on sucrose gradients, stability of the hormone-receptor complex at 0 degrees C, and the retention patterns on phosphocellulose and DEAE cellulose. Multiple forms of the receptor exist in a single homogeneous cell type. The data support the theory that steroid hormones bind to a cytoplasmic protein receptor composed of dissimilar subunits as the initial step in steroid hormone action.

International Society for Cell and Gene Therapy of Cancer: 2005 meeting in Shenzhen, China.

The 2005 International Society for Cell and Gene Therapy of Cancer (ISCGT) Congress was held in Shenzhen, China (www.iscgtchina2005.com) from December 9th-11th 2005. Here, we describe a representation of the most seminal presentations providing an overview of the progress in the field of cancer gene therapy including the successful introduction of the first approved gene therapy drug.

FasL gene therapy: a new therapeutic modality for head and neck cancer.

In this study, we investigated the in vitro and in vivo efficacy of Fas ligand (FasL) gene therapy for the treatment of head and neck cancer. Three head and neck squamous cell carcinoma (HNSCC) cell lines (SCC-1, SCC-12, and SCC-14a) were treated with the Fas agonist CH-11, a monoclonal antibody to the Fas receptor, or with a replication-incompetent adenovirus (AdGFPFasL) expressing a modified murine Fas ligand gene fused to green fluorescent protein (GFP). A replication-incompetent adenovirus containing the GFP gene alone was used as a control for viral transduction toxicity (AdGFP). Cell death was quantified using a tetrazolium-based (MTS) assay. Cells were analyzed by flow cytometry to determine the expression of adenoviral and Fas receptors on the surface of the cells. Our results showed that the head and neck cancer cell lines are resistant to cell death induction when treated with the anti-Fas monoclonal antibody CH-11. This resistance can be overcome with AdGFPFasL, which was able to induce cell death in all three cell lines. Apoptosis induction was demonstrated using Western blotting by evaluating poly(ADP-ribose) polymerase, and caspase 9 cleavages. In addition, intratumoral injections of AdGFPFasL into SCC-14a xenografts induced significant growth suppression of tumors, indicating that FasL gene therapy may provide a new efficient therapeutic modality for HNSCC that is worthy of a clinical trial.

Combined therapeutic use of AdGFPFasL and small molecule inhibitors of ceramide metabolism in prostate and head and neck cancers: a status report.

As of January 2005, there were 1020 gene therapy clinical trials ongoing worldwide with 675 or 66.2% devoted to cancer gene therapy. The majority are occurring in the US and Europe (http://www.wiley.co.uk/genetherapy/clinical/). At the present time, to our knowledge there are no trials that employ gene delivery of Fas Ligand (FasL). As an important note, and in contrast to somatic cell therapy trials, there are no reported deaths due to therapeutic vector administration in any cancer gene therapy trial. That said, from our studies and from the published literature, the issue of gene delivery remains the major obstacle to successfully employing gene therapy for cancer treatment. Numerous laboratories are studying this with many different approaches. My co-workers and I have focused on the delivery issue by using various approaches that address tumor targeting and transgene expression. In addition, we are focusing on enhancing tumor cell killing via the bystander effect and through use of small molecules to enhance bystander activity.

Isolation and characterization of a cloned cell line R3327H-G8-A1 derived from the dunning R3327H rat adenocarcinoma.

A cloned cell line (R3327H-G8-A1) has been isolated from the Dunning R3327H adenocarcinoma. Light and electron microscopic studies showed that the cell line possessed features common to secretory epithelial cells. These cells, which grow in monolayer culture, produced s.c. hind flank tumors when inoculated inoculated into Copenhagen X Fischer F1 rats.l Chromosomal karyotype analysis confirmed that the cell line is distinctly that of the Rattus norvegicus genus and species. The cells specifically bind testosterone and dexamethasone with equilibrium dissociation constants (Kd) of 0.49 and 0.8 nM, respectively. The numbers of saturable binding sites per cell are 10,000 for testosterone and 60,000 for dexamethasone. The cells also have 5 alpha-reductase activity. These properties are characteristic of the prostate and of the Dunning tumor from which the cells are derived. Cell growth in vitro was stimulated by androgens and inhibited by glucocorticoids at concentrations of 10(-8) M. An int riguing finding was that estradiol and progestins dramatically stimulated growth in the apparent absence of receptors for these hormones. Finally, comparisons between the G8-A1 cells and the tumor induced by the G8-A1 clone and a second generation of cells from this G8-A1-induced tumor showed that the cloned cells retained their properties following passaging in the animal.

Nuclear factor-kappaB/IkappaB signaling pathway may contribute to the mediation of paclitaxel-induced apoptosis in solid tumor cells.

Paclitaxel (Taxol), a naturally occurring antimitotic agent, has shown significant cell-killing activity in a variety of tumor cells through induction of apoptosis. The mechanism by which paclitaxel induces cell death is not entirely clear. Recent studies in our laboratory demonstrated that glucocorticoids selectively inhibited paclitaxel-induced apoptosis without affecting the ability of paclitaxel to induce microtubule bundling and mitotic arrest. This finding suggests that apoptotic cell death induced by paclitaxel may occur via a pathway independent of mitotic arrest. In the current study, through analyses of a number of apoptosis-associated genes or regulatory proteins, we discovered that paclitaxel significantly down-regulated IkappaB-alpha, the cytoplasmic inhibitor of transcription factor nuclear factor-kappaB (NF-kappaB), which in turn promoted the nuclear translocation of NF-kappaB and its DNA binding activity. In contrast, we found that glucocorticoids could antagonize paclitaxel-mediated NF-kappaB nuclear translocation and activation through induction of IkappaB-alpha protein synthesis. Northern blotting analyses demonstrated that the steady-state level of IkappaB-alpha mRNA was not affected by paclitaxel, which suggests that the down-regulation of IkappaB-alpha by paclitaxel is attributable to protein degradation rather than suppression of transcription. Furthermore, through transfection assays, we demonstrated that tumor cells stably transfected with antisense IkappaB-alpha expression vectors remarkably increased their sensitivity to paclitaxel-induced apoptosis. Finally, we found that a key subunit of IkappaB kinase (IKK) complex, IKKbeta, was up-regulated by paclitaxel, which implies that paclitaxel might down-regulate IkappaB-alpha through modulation of IKKbeta activity. All of these results suggest that the NF-kappaB/IkappaB-alpha signaling pathway may contribute to the mediation of paclitaxel-induced cell death in solid tumor cells.

Overexpression and amplification of c-myc in the Syrian hamster kidney during estrogen carcinogenesis: a probable critical role in neoplastic transformation.

An estrogen receptor-driven, multistep process for estrogen carcinogenesis in the Syrian hamster kidney is proposed. Because in this species the reproductive and urogenital tracts arise from the same embryonic germinal ridge, it is evident that the kidney has carried over genes that are responsive to estrogens. Using in situ hybridization, overexpression of early estrogen-response genes, i.e., c-myc and c-fos, has been shown to be localized preferentially in early renal tumor foci after 3.5-4.0 months of estrogen treatment. This event coincides with an increased number of S-phase proliferating cell nuclear antigen-labeled cells in these tumor foci, along with a rapid rise in aneuploid frequency in the kidney. Western blot analyses of c-MYC and c-FOS protein products support the overexpression of these genes. Amplification of c-myc, 2.4-3.6-fold, but not of c-fos, was detected in 67% of the primary renal tumors examined, by Southern blot analyses. Consistent chromosomal gains, common to both diethylstilbestrol- and estradiol-induced renal neoplasms, were observed in chromosomes 1, 2, 3, (6), 11, (13), 16, 20, and 21 (chromosome number alterations are indicated in parentheses). Using fluorescence in situ hybridization, the c-myc gene was localized to hamster chromosome 6qb. Chromosome 6 exhibited a high frequency of trisomies and tetrasomies in the kidney after 5.0 months of estrogen treatment and in primary renal tumors. The data presented indicate that estrogen-induced genomic instability may be a key element in carcinogenic processes induced by estrogens.

Molecular cloning of TA16, a transcriptional repressor that may mediate glucocorticoid-induced growth arrest of leiomyosarcoma cells.

The DDT1 MF2 smooth muscle tumor cell line was derived from an estrogen/androgen-induced leiomyosarcoma that arose in the ductus deferens of a Syrian hamster. The growth of this cell line is arrested at the G0/G1 phase of the cell cycle after treatment with glucocorticoids. To identify the putative gene(s) that are potentially involved in this hormone-induced cell growth arrest, we have used a differential screening technique to clone those genes whose expression is induced or up-regulated by glucocorticoids. A number of glucocorticoid response genes were thereby isolated from the leiomyosarcoma cells. One of these clones, termed TA16, was found to be markedly up-regulated by glucocorticoids in DDT1 MF2 cells, but only marginally changed in GR1 cells, a glucocorticoid-resistant variant that was selected from the wild type DDT1 MF2 cell. Isolation and sequencing of its intact cDNA indicated that the TA16 encodes a protein 485 amino acids long, and its sequence is closely homologous to a novel transcriptional repressor that presumably represses the transcription activity of some zinc finger transcriptional factors through a direct interaction. Transfection assays demonstrated that introduction of an antisense TA16 cDNA expression vector, controlled by an MMTV promoter, into the DDT1 MF2 cell significantly relieved the glucocorticoid-induced cell growth arrest. This finding suggests that TA16 might participate in the mediation of glucocorticoid-induced cell cycle arrest in leiomyosarcoma cells.

Cloning of a mu-class glutathione S-transferase complementary DNA and characterization of its glucocorticoid inducibility in a smooth muscle tumor cell line.

A cDNA (designated hGSTYBX) encompassing the complete coding sequence of a hamster mu-class glutathione S-transferase (GST) subunit was cloned from a lambda ZAP library constructed with mRNA isolated from triamcinolone acetonide-treated smooth muscle tumor cells (DDT1 MF-2). Analysis of its nucleotide and deduced amino acid sequences demonstrated highest homology to the rat mu-class GST YB2 subunit. In proliferating subconfluent cells, in which constitutive expression of hGSTYBX mRNA was undetectable, glucocorticoid treatment induced hGSTYBX expression after a time lag of 3 h, and maximal induction occurred at 10 h. Nuclear run-on analysis showed that glucocorticoid induction resulted at least in part from an increased rate of transcription. Simultaneous treatment with glucocorticoid and cycloheximide prevented glucocorticoid induction, but had little effect on basal expression in confluent cells. In contrast, cycloheximide treatment 3 h after glucocorticoid treatment resulted in nearly full induction. These results taken together suggest that hGSTYBX induction may be a secondary glucocorticoid response.

Aneurysmal subarachnoid haemorrhage: guidance in making the correct diagnosis.

BACKGROUND: The natural history of untreated aneurysmal subarachnoid haemorrhage carries a dismal prognosis. Case fatalities range between 32% and 67%. Treatment with either surgical clipping or endovascular coiling is highly successful at preventing re-bleeding and yet the diagnosis is still missed. METHODS: Based on the national guidelines for analysis of cerebrospinal fluid for bilirubin in suspected subarachnoid haemorrhage and a review of other available literature this study has compiled guidance in making the diagnosis. CONCLUSION: In patients presenting with a suspected non-traumatic subarachnoid haemorrhage, computed tomography within 12 hours will reliably show 98% of subarachnoid haemorrhage. In patients who present after 12 hours with a negative computed tomogram, formal cerebrospinal fluid spectophotometry will detect subarachnoid haemorrhage for the next two weeks with a reliability of 96%. Between the early diagnosis with the aid of computed tomography and the later diagnosis with the added benefit of spectophotometry in the period where computed tomograms become less reliable, it should be possible to diagnose most cases of subarachnoid haemorrhage correctly.

The coexistence of androgen and glucocorticoid receptors in the DDT1 cloned cell line.

The hamster ductus deferens cloned tumor cell line (DDT1) has been shown to contain both androgen and glucocorticoid binding activity. The androgen receptor binding site concentration is 1.07 x 10(-13) mol of testosterone/mg protein, and testosterone (T) binds with a Kd of 4.3 x 10(-10) M. Dihydrotestosterone (DHT) is also bound to the receptor with a Kd of 2.99 x 10(-10) M and the binding site concentration is 1.33 x 10(-13) mol/mg protein. The order of steroid binding affinity is DHT greater than T greater than Estradiol greater than Progesterone. Cortisol, dexamethasone, and triamcinolone acetonide do not inhibit the androgen binding in vivo or in vitro. In a cell free system antiandrogens inhibit the binding of DHT. The DDT1 cells have a separate receptor for cortisol which binds at saturation 3.44 x 10(-13) mol cortisol/mg protein and has a Kd of 4.54 x 10(-9) M. These studies provide evidence that these endocrine target cells contain specific high affinity receptors for more than one type of steroid. The glucocorticoid receptor may be important for maintaining essential undifferentiated functions while the DHT receptor gives the specific characteristics of sex hormone responsive tissues.

Construction of a retrotransposition indicator sequence using a neomycin resistance-encoding gene containing a functional intron.

An intron from a Syrian hamster gene was inserted into a neo gene such that splicing of the neo gene mRNA results in the synthesis of active aminoglycoside phosphotransferase. The unspliced construct is inactive in Escherichia coli, but confers resistance to G418 when transfected into mouse and hamster cells. This selectable marker is designed to aid in the cloning and identification of genomic integration sites following retrotransposition.

The use of Fas Ligand, TRAIL and Bax in gene therapy of prostate cancer.

Prostate cancer is the second leading cause of cancer death in the United States. Treatment options for confined disease are generally successful in prolonging life but long-term cures (10-15 years) are elusive for the majority of patients. The prognosis for advanced extra-capsular prostate cancer is grim. However, we are now entering the era of gene therapy options for treatment of prostate cancer. The human genome project coupled with genomics and protemics are providing information that will lead to selection of genes for treatment of prostate cancer. The problem is the science of delivery lags behind knowledge of gene function. Thus, it is important to develop therapies that do not require delivery to 100% of tumor cells but which nevertheless kills the entire cancer by virtue of the bystander effect or other means. This review covers the use, in gene therapy, of apoptotic inducing molecules such as Fas Ligand, and TRAIL which are believed to induce bystander killing activity and Bax which also may function in a similar way.

Exogenous 17beta-estradiol blocks alpha and mu but not pi class glutathione S-transferase immunoreactivity in epithelium of Syrian hamster vas deferens.

Members of the glutathione S-transferase (GST) family of detoxification enzymes play a role in chemotherapy resistance in certain cancers but have not been directly implicated as agents whose absence may predispose tissues to hormonally induced tumorigenesis. Here we report the development of a polyclonal antiserum to a hamster mu class GST, and immunohistochemical analysis of alpha, mu, and pi class GSTs to study the effects of hormone treatment on their expression in reproductive tract tissues of male golden Syrian hamsters. These animals develop leiomyosarcomas in the vas deferens after treatment with testosterone propionate (TP) and 17beta-estradiol (E2). High levels of all three GST classes were detected throughout the reproductive tract epithelium of control animals. In 100% of the experimental animals, 4 weeks of treatment either with E2 alone, or with E2 plus TP promoted a complete loss of immunostaining for alpha and mu class GSTs, but not for pi class GSTs, only in the epithelial lining of the vas deferens. In contrast, treatment with TP alone resulted in moderate hyperplasia of smooth muscle in the proximal vas deferens, with no cellular atypia and no changes in immunoreactivity of any of the GST classes. The consistent and site-specific nature of these results strongly suggests a functional role for GSTs in hormonally induced carcinogenic process. (J Histochem Cytochem 47:91-98, 1999)

Affinity labeling of the DDT1 MF-2 cell alpha 1-adrenergic receptor with [3H]phenoxybenzamine.

In this study, we used phenoxybenzamine to label the alpha 1-adrenergic receptor of a smooth muscle cell line. Our results demonstrate a dose-dependent occupancy of alpha 1-adrenergic receptors by phenoxybenzamine determined by competition for the [3H]prazosin binding site. Following incorporation of [3H]phenoxybenzamine, partially purified membranes were solubilized and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Despite numerous Coomassie blue-stained bands, only three bands, Mr = 80,000 +/- 500, Mr = 33,000 +/- 2,000, and Mr = 21,000 +/- 400 (N = 4), were labeled with [3H]phenoxybenzamine as determined by autofluorography. Incorporation of [3H]phenoxybenzamine into the Mr = 80,000 band, but not the Mr = 33,000 and Mr = 21,000 bands, was affected by adrenergic agonists and antagonists in a manner consistent with an alpha 1-adrenergic interaction. Labeling of the Mr = 33,000 and Mr = 21,000 bands was partially blocked by phenoxybenzamine. We conclude that [3H]phenoxybenzamine can be used as an affinity probe for the alpha 1-adrenergic receptor and that the ligand binding site of the alpha 1-adrenergic receptor resides in a Mr = 80,000 protein.

Specific binding sites for prohormone atrial natriuretic peptides 1-30, 31-67 and 99-126.

Two peptides with vasodilatory properties consisting of amino acids 1-30 and 31-67 of the 98 a.a. N-terminal end of the prohormone of atrial natriuretic factor (proANF) which circulates in man were investigated to determine if they have specific binding sites on membranes isolated from DDT1 MF-2 smooth muscle cells. Smooth muscle is a known biologic target of these peptides. Competitive binding experiments revealed that proANFs (1-30), (31-67), and (99-126) (i.e., C-terminus; ANF) each had specific and separate binding sites. The dissociation constants for proANFs (1-30), (31-67), and (99-126) binding were 0.11 nM, 4 nM, and 7.3 nM, respectively. The binding site concentrations for proANFs (1-30), (31-67), and ANF were 2.57, 59.91 and 40 fmols/10(6) cells, respectively. The number of binding sites per cell were 1548, 36,087, and 24,090, respectively, for proANFs (1-30), (31-67), and (99-126) (ANF). Each peptide bound to DDT1 MF-2 membranes between 10(-8) to 10(-11) M but could only bind to the other peptides' receptors at concentrations of 10(-6) and 10(-7)M. These results suggest that proANF(1-30) and proANF(31-67) do not work through the ANF receptor but rather have their own separate and distinct receptors that mediate their biologic effects.

Gliosarcoma with chromosome abnormalities in a neonate exposed to heptachlor.

A neonate with a cerebral gliosarcoma was found to have chromosome abnormalities in tissue culture of the tumor, but normal karyotyping of peripheral blood. Similarities to and differences from chromosome abnormalities found in other human gliomas are noted. Unusual exposure of the child to heptachlor during prenatal development and the neonatal period suggests the need for further studies on the role of toxins in oncogenesis.

Prospective study of zero drift in fiberoptic pressure monitors used in clinical practice.

One hundred and one fiberoptic pressure transducers (59 subdural and 42 ventricular) were studied in 86 patients (some in whom more than one device had been inserted). Only four complications occurred: two transient cerebrospinal fluid leaks after removal and two clinically insignificant intracerebral hematomas. No intracranial infections could be attributed to the devices. Technical problems occurred 23 times, with 11 devices ceasing to function before removal, seven becoming displaced, and five microventricular catheters failing to enter the ventricles. Zero-drift readings were obtained for 83 devices at the time of removal (median 66 hours after insertion, range 2 hours-13 days). There was a clear negative bias in the readings (median -3), with a wide range of values (-12 to +14 mm Hg; interquartile range -6 to -1) that was apparent even in the first 3 days of use. There was no important relationship between zero drift and any recorded variable. It is concluded that zero drift of fiberoptic pressure transducers is a significant problem and that undue reliance should not be placed on intracranial pressure readings from these devices in isolation from other clinical and radiological information.

A simple relationship between radiological arteriovenous malformation hemodynamics and clinical presentation: a prospective, blinded analysis of 31 cases.

OBJECT: The authors sought to establish prospectively whether there is a simple relationship between radiological features of brain arteriovenous malformation (AVM) hemodynamics and a patient's clinical presentation. METHODS: Thirty-one consecutive patients with AVMs underwent cerebral angiography at 3.8 frames/second during each standardized injection of contrast material. Contrast dilution curves were derived from the image sequences by using regions of interest (ROIs) traced on arteries feeding and veins draining the AVM nidus. Angiographic parameters were then analyzed in a blinded fashion. These parameters included the times required to reach the peak contrast density, the contrast decay time, and fractions thereof, in the ROI for each vessel. The authors determined whether these parameters, the arteriovenous transit time, and/or AVM size were related to patients' presentation with hemorrhage (11 patients), seizure (11 patients), or other clinical symptoms (nine patients). Statistically significant results were found only in analyses of arterial phase times to reach peak contrast density. Analyses of venous parameters, AVM size, and nidus transit time showed trends but no statistical significance. Arterial filling with contrast material was significantly slower in patients presenting with hemorrhage (mean 50%, 80%, and 100% of time to peak +/- standard error [SE] = 1.19+/-0.13, 1.97+/-0.18, and 3.04+/-0.34 seconds, respectively) compared with patients presenting with seizures (mean 50%, 80%, and 100% of time to peak +/- SE = 0.80+/-0.12, 1.32+/-0.18, and 1.95+/-0.29 seconds, respectively) according to analysis of variance (p<0.05) and post-hoc t-tests (p<0.05) for each parameter. Patients who presented with other symptoms had intermediate arterial filling times. CONCLUSIONS: These simple hemodynamic parameters, which can be obtained without added risk to the patient, may help identify a subset of individuals in whom AVMs pose a higher risk of future hemorrhage and who may therefore warrant more expeditious treatment.

Blockade of alpha-adrenergic receptors by analogues of phosphatidylcholine.

The experimental evidence reviewed in this article suggests that the kidneys may have an additional function in regulating blood pressure besides their role in controlling both blood volume by urine formation and the relative state of vasoconstriction by the renin-angiotensin system. That is, the kidneys may have an additional influence upon the vasculature of a hormonal vasodilating system. The interstitial cells of the renal medulla appear to be mediating this activity and lipid compounds have been extracted from the renal medulla which display depressor activity. One such compound, the antihypertensive polar renomedullary lipid (APRL), has been demonstrated to consist of specific alkyl ether analogues of phosphatidylcholine. The vascular responses to these compounds include vasodilation of both arterioles and venules, rapid lowering of arterial blood pressure with little or no tachycardia, increased depressor activity in hypertensive animals, and blockade of vascular smooth muscle alpha 1-adrenergic receptors. Most recently, APRL and a synthetic analogue, 1-0-octadecyl-2-acetyl-sn-glycero-3-phosphorylcholine, have been used to demonstrate alpha-adrenergic receptor blockade on a smooth muscle cell line (DDT1) by radioligand assays. This action may be due to the insertion of these compounds into cell membranes causing subsequent steric interactions and blockade of the alpha-adrenergic receptor.