The epidemic of human papillomavirus and oropharyngeal cancer in a Canadian population.

BACKGROUND: Sexually transmitted infection with the human papillomavirus (hpv) is responsible for a significant burden of human cancers involving the cervix, anogenital tract, and oropharynx. Studies in the United States and Europe have demonstrated an alarming increase in the frequency of hpv-positive oropharyngeal cancer, but the same direct evidence does not exist in Canada. METHODS: Using the London Health Sciences Centre pathology database, we identified tonsillar cancers diagnosed between 1993 and 2011. Real-time polymerase chain reaction was then used on pre-treatment primary-site biopsy samples to test for dna from the high-risk hpv types 16 and 18. The study cohort was divided into three time periods: 1993-1999, 2000-2005, and 2006-2011. RESULTS: Of 160 tumour samples identified, 91 (57%) were positive for hpv 16. The total number of tonsillar cancers significantly increased from 1993-1999 to 2006-2011 (32 vs. 68), and the proportion of cases that were hpv-positive substantially increased (25% vs. 62%, p < 0.002). Those changes were associated with a marked improvement in 5-year overall survival (39% in 1993-1999 vs. 84% in 2006-2011, p < 0.001). When all factors were included in a multivariable model, only hpv status predicted treatment outcome. INTERPRETATION: The present study is the first to provide direct evidence that hpv-related oropharyngeal cancer is increasing in incidence in a Canadian population. Given the long lag time between hpv infection and clinically apparent malignancy, oropharyngeal cancer will be a significant clinical problem for the foreseeable future despite vaccination efforts.

Combining small interfering RNAs targeting thymidylate synthase and thymidine kinase 1 or 2 sensitizes human tumor cells to 5-fluorodeoxyuridine and pemetrexed.

Thymidylate synthase (TS) is the only de novo source of thymidylate (dTMP) for DNA synthesis and repair. Drugs targeting TS protein are a mainstay in cancer treatment, but off-target effects and toxicity limit their use. Cytosolic thymidine kinase (TK1) and mitochondrial thymidine kinase (TK2) contribute to an alternative dTMP-producing pathway, by salvaging thymidine from the tumor milieu, and may modulate resistance to TS-targeting drugs. Combined down-regulation of these enzymes is an attractive strategy to enhance cancer therapy. We have shown previously that antisense-targeting TS enhanced tumor cell sensitivity to TS-targeting drugs in vitro and in vivo. Because both TS and TKs contribute to increased cellular dTMP, we hypothesized that TKs mediate resistance to the capacity of TS small interfering RNA (siRNA) to sensitize tumor cells to TS-targeting anticancer drugs. We assessed the effects of targeting TK1 or TK2 with siRNA alone and in combination with siRNA targeting TS and/or TS-protein targeting drugs on tumor cell proliferation. Down-regulation of TK with siRNA enhanced the capacity of TS siRNA to sensitize tumor cells to traditional TS protein-targeting drugs [5-fluorodeoxyuridine (5FUdR) and pemetrexed]. The sensitization was greater than that observed in response to any siRNA used alone and was specific to drugs targeting TS. Up-regulation of TK1 in response to combined 5FUdR and TS siRNA suggests that TK knockdown may be therapeutically useful in combination with these agents. TKs may be useful targets for cancer therapy when combined with molecules targeting TS mRNA and TS protein.

Rodent PSP94 gene expression is more specific to the dorsolateral prostate and less sensitive to androgen ablation than probasin.

To date, the rodent ventral prostate (VP) has been the focus of many studies on androgen action, less attention has been directed to the lateral prostate (LP) and the dorsal prostate (DP). The rodent VP has no clear homologous counterpart in the human prostate. The rodent LP and DP is the only prostate lobe comparable to the peripheral zone of the human prostate, where hormone-induced prostate cancer mainly occurs. To explore its utility for prostate targeting, we have studied the gene expression of PSP94 with rat probasin (rPB), a gene commonly used for prostate targeting in prostate cancer research and a gene typically responsive to androgen regulation. Firstly, we demonstrated PSP94 gene transcription being more specific to the LP and DP lobes than rPB, where rPB RNA was detected in the LP and DP and other lobes at different levels. Secondly, we found that PSP94 gene transcription decreased relatively slowly in response to androgen deprivation but recovered rapidly in response to testosterone replacement after complete ablation of PSP94 transcription. In the VP, gene transcripts of rPB were specifically responsive to androgen deprivation; however, they responded relatively slowly in the LP and DP. RNase protection experiments indicated that the slow response was not due to abnormal persistence of PSP94 messenger RNA specifically in the DP and LP lobes in comparison with rPB. Thirdly, Western blot analysis revealed that both PSP94 and rPB expression is specific to the LP and DP at the protein level, exhibiting slow responses to testosterone replacement after castration. We conclude that PSP94 gene expression at the transcriptional level is more specific to the LP and DP than rPB and thus less sensitive to androgen ablation. This may have clinical implications for strategies to target the prostate in cancer therapy.

Effect of non-toxic mercury, zinc or cadmium pretreatment on the capacity of human monocytes to undergo lipopolysaccharide-induced activation.

1. Metal salts can inhibit cell activity through direct toxicity to critical cellular molecules and structures. On the other hand, they can also change cell behaviour by inducing specific genes (including genes encoding members of the metallothionein [MT] gene family). Therefore, transition metals may affect cell functions either by acting as a toxin, or by transmitting or influencing signals controlling gene expression. 2. To explore the latter possibility, we measured the ability of low, non-toxic metal pretreatment to alter immune cell behaviour. We previously found that pretreatment of human monocytes with zinc induces metallothionein gene expression and alters their capacity to undergo a bacterial lipopolysaccharide-induced respiratory burst. We showed here that cadmium and mercury salts, at concentrations that exert no discernible toxicity, inhibit activation of human monocytic leukemia (THP-1) cells. CdCl2 1 microM, ZnCl2 20-40 microM or HgCl2 2 microM pretreatment for 20 h induced MT-2 mRNA and total MT protein accumulation and had no effect on proliferation potential or metabolic activity, but significantly inhibited the ability of subsequent lipopolysaccharide treatment to induce the oxidative burst, increased adhesion to plastic, and MT-2 and interleukin-1 beta (IL-1 beta) mRNA accumulation. 3. The phenomenon of metal-induced suppression of monocyte activation, at metal concentrations that have no effect on cell viability, has important implications for assessment of acceptable levels of human exposure to cadmium, zinc and mercury.

Antisense-induced down-regulation of thymidylate synthase and enhanced cytotoxicity of 5-FUdR in 5-FUdR-resistant HeLa cells.

1. Thymidylate synthase (TS) is a target for several anticancer drugs. We previously showed that an antisense oligodeoxynucleotide (ODN) directed against TS mRNA down-regulated TS protein and enhanced cytotoxicity of TS-targeting drugs [including 5-fluorodeoxyuridine (5-FUdR)] in HeLa cells. Patient tumours with increased TS expression are resistant to TS-targeting drugs. It was hypothesized that TS mRNA and consequently TS protein could be down-regulated in 5-FUdR-resistant cells that overexpress TS, sensitizing them to 5-FUdR cytotoxicity. In this study we assessed the capacity of an anti-TS antisense ODN to circumvent resistance dependent on TS overexpression. 2. Variant HeLa clones exhibiting 2 - 20 fold resistance to 5-FUdR were selected by exposing cultured cells to drug. Clones FUdR-5a, -25b, and -50a expressed TS protein levels 10 fold, 10 fold, and 17 fold higher (respectively) than parental cells. Cells were treated with antisense ODN 83 (a 2'-methoxy-ethoxylated, phosphorothioated 20-mer, complementary to a portion of the 3'-untranslated region of TS mRNA), or ODN 32 (a control ODN with the same base composition as ODN 83, but in randomized order). Twenty-four and 48 h following transfection (50-100 nM ODN, plus polycationic liposome), TS mRNA levels (by RT-PCR) and protein levels (by radiolabelled 5-FUdR-monophosphate binding) were decreased by at least 60% in ODN 83-treated cells compared with control ODN 32-treated cells. ODN 83 enhanced the cytotoxicity of 5-FUdR by up to 85% in both parental and 5-FUdR-resistant cell lines. 3. Antisense ODN can be used to down-regulate TS and attenuate drug resistance in TS-overexpressing cells.

Antisense down-regulation of thymidylate synthase to suppress growth and enhance cytotoxicity of 5-FUdR, 5-FU and Tomudex in HeLa cells.

1. Thymidylate synthase (TS), the key enzyme in de novo synthesis of thymidine, is an important target for antitumour chemotherapy. It was hypothesized that antisense oligonucleotide down-regulation of TS mRNA would decrease TS levels and enhance the cytotoxicity of inhibitors of TS, including the pyrimidine analogues 5-fluorouracil (5-FU) and 5-fluorodeoxyuridine (5-FUdR), and the folate analogue Tomudex (ICI D1694; N-(5-[N-(3, 4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N-methylamino ]-2-theon yl-L-glutamic acid). 2. 2'-Methoxyethoxylated, phosphorothioated 20-mer oligodeoxynucleotides (ODNs), complementary to various sequences in TS mRNA, were synthesized, along with control oligomers consisting of the same, respective bases in randomized order, against which all the biological effects were compared. Following a 6-h transfection of HeLa cells using polycationic liposome at 3 microg ml(-1), ODN 83 (50 nM), complementary to a region in the 3'-untranslated region of the TS mRNA, decreased TS mRNA levels by approximately 70% within 24 h. ODN 83 also decreased TS enzyme activity, as measured by binding of TS to radiolabelled 5-fluorodeoxyuridine monophosphate. In addition to inhibiting proliferation by up to approximately 40%, ODN 83 enhanced the cytotoxicity of Tomudex or 5-FU, added 1 day following transfection, by 50 - 60%. ODN 83 also enhanced sensitivity to 5-FUdR by 70%, but did not affect the toxicity of cisplatin, chlorambucil, melphalan, doxorubicin, ionizing radiation, paclitaxel, or irinotecan. 3. These data indicate that antisense ODN down-regulation of TS can inhibit human tumour cell proliferation and enhance the efficacy of TS-targeted drugs.

Organ-specific metallothionein induction in mice by X irradiation.

Metallothioneins (MTs) are induced in cultured cells and experimental animal tissues by a variety of chemical agents. We report that whole-body X irradiation (1 to 80 Gy) induces MT-1 mRNA transcription and protein expression and accumulation in liver but not in kidney or spleen. The degree of induction was comparable to maximum levels achieved after treatment with metal salts, but the peak of MT-1 mRNA and protein accumulation was approximately 10 h later than with treatment with metal salts and remained high for an extended period. Because of the lack of induction of MT-1 by X rays in cultured cells, and the similarity of the tissue pattern of MT induction between X rays and other agents that also do not induce MT expression in cultured cells, it appears that these agents may act through the mediation of tissue-specific factors. The implications of radiation-induced metallothionein synthesis and organ-specific resistance to cellular damage are discussed.

Zinc-metallothionein levels are correlated with enhanced glucocorticoid responsiveness in mouse cells exposed to ZnCl(2), HgCl(2), and heat shock.

Metallothioneins (MTs) are the major low molecular weight, zinc-binding proteins in mammalian cells. It has been hypothesized that they play a role in the function of zinc-dependent signal transduction proteins and transcription factors. We investigated the capacity of zinc and other metal ions and conditions to increase both Zn-associated MT levels and the receptiveness of cells to transcriptional activation mediated by the zinc-dependent glucocorticoid receptor (GR). We studied, in a GR-responsive mouse mammary-tumor cell line, the ability of dexamethasone (DEX) to stimulate transcription of a chloramphenicol acetyltransferase (CAT) gene controlled by a mouse mammary-tumor virus promoter. In cells pretreated with 20 to 100 microM ZnCl(2), DEX-induced CAT activity correlated with zinc-induced MT levels. However, 0.05 to 0.5 microM CdCl(2) had no effect on CAT activity, despite an increase in Cd-associated MT. Copper-associated MT was detected in cells treated with 20 microM CuCl(2,) but there was no change in the level of Zn-MT, nor was CAT activity altered in cells exposed to 5 to 20 microM CuCl(2). These results may reflect a functional difference between zinc-associated MT, and MT associated with other metals. Significantly more CAT activity was observed in both heat-shocked cells and in cells exposed to 40 or 50 nM HgCl(2). Although absolute amounts of MT were unchanged by these two treatments, a higher percentage of total cellular zinc was associated with the MT protein fractions after treatment. Changes in GR levels could not account for variations in CAT activity. These data indicate that hormonal signalling can be altered by exposure to metal salts and heat shock, and the effect is correlated with the level of Zn-MT.

Induction of metallothionein synthesis by zinc in cadmium pretreated rats.

The ability of zinc (Zn) salts to induce the synthesis of metallothionein (MT) in liver, kidney and pancreas of rats pretreated with cadmium (Cd) salts was investigated. Twenty-four hours after either CdCl2 (2.0 mg Cd/kg, s.c.) or saline pretreatment, rats were injected with saline, CdCl2 (2.0 mg Cd/kg, s.c.) or ZnSO4 (20 mg Zn/kg, s.c.) and the concentrations of MT and MT-1 mRNA in tissues subsequently measured. After a single injection of Cd salts, concentrations of MT and MT-1 mRNA were significantly increased in liver as compared to control. With two injections of Cd, the accumulation of MT in liver was approximately twice the levels of MT following a single injection of Cd. In kidney, MT and MT-1 mRNA expression were significantly increased only after two injections of Cd and in the pancreas, Cd injections did not alter either MT content or MT-1 mRNA expression. Treatment with Zn salts increased MT concentrations in both liver and pancreas. However, the pancreas was the most responsive to injections of Zn salts as compared to the liver in terms of increases in both protein concentration and MT-1 mRNA expression. When Zn injection was preceded by a Cd injection, induction as measured by MT-1 mRNA and MT concentrations were approximately additive in liver. In kidney, although Cd or Zn treatment separately had no effect on MT or MT-1 mRNA content, injection of Cd followed by Zn resulted in significantly increased levels of renal MT and MT-1 mRNA. Fractionation of liver cytosols on a Sephadex G-75 column revealed that in animals receiving two injections of Cd, virtually all the Cd was associated with MT whereas Zn was distributed between both high molecular weight (HMW) proteins and MT. In animals receiving both Cd and Zn injections, cytosolic Cd was still bound predominantly to the MT fraction, while the proportion of cytosolic Zn associated with MT increased. The results of this study suggest that, treatment with Cd salts followed by Zn salt injection can induce further synthesis of MT in liver, kidney and pancreas with subsequent binding of both Zn and Cd to the intracellular MT.

Metallothionein protects DNA from copper-induced but not iron-induced cleavage in vitro.

Iron and copper ions mediate generation of reactive oxygen radicals from O2 and H2O2 by the Fenton reaction: these radicals are capable of damaging DNA. We studied (a) the ability of these metals to induce double-strand breaks in DNA in vitro in the presence of H2O2 and ascorbic acid as donors of reactive oxygen, and (b) the ability of the metal-binding protein metallothionein (MT) to protect DNA from damage. Strand cleavage was measured by loss of fluorescence after binding to ethidium bromide and by increased mobility of DNA in agarose. The results show that Cu(II), Fe(II) and Fe(III) all can induce damage to calf thymus DNA under our experimental conditions. Cu(II)-induced DNA damage was dose-dependent and the degree of damage was proportional to the concentration of H2O2. On the other hand, DNA fragmentation was significant only in the presence of high concentrations of Fe(II) or Fe(III). Addition of Zn-MT to the reaction mixture prior to addition of Cu(II) inhibited fragmentation of DNA in a dose-dependent manner but had little effect on iron induced damage. Other proteins (histone or albumin) were not effective in protecting DNA from Cu-induced damage, as compared to Zn-MT. The formation of Cu(I) from Cu(II) in the presence of hydrogen peroxide and ascorbate was also inhibited by addition of Zn-MT. Thus, MT may protect DNA from damage by free radicals by sequestering copper and preventing its participation in redox reactions.

Roles of vitamin C in radiation-induced DNA damage in presence and absence of copper.

Exposure to either ionizing radiation or certain transition metals results in generation of reactive oxygen species that induce DNA damage, mutation, and cancer. Vitamin C (a reactive oxygen scavenger) is considered to be a dietary radioprotective agent. However, it has been reported to be genotoxic in the presence of certain transition metals, including copper. In order to explore the capacity of vitamin C to protect DNA from radiation-induced damage, and the influence of the presence of copper on this protection, we investigated vitamin C-mediated protection against radiation-induced damage to calf thymus DNA in vitro in the presence or absence of copper(II). Vitamin C (0.08-8.00 mM, pH 7.0) significantly reduced DNA damage induced by gamma-irradiation (30-150 Gy) by 30-50%, similar to the protective effect of glutathione. However, vitamin C plus copper (50 microM) significantly enhanced gamma-radiation-induced DNA damage. Low levels of added copper (5 microM), or chelation of copper with 1-N-benzyltriethylenetetraine tetrahydrochloride (BzTrien) and bathocuprinedisulfonic acid (BCSA), abolished the enhanced damage without diminishing the protective effect of vitamin C. These results indicate that vitamin C can act as: (1) an antioxidant to protect DNA damage from ionizing radiation; and (2) a reducing agent in the presence of copper to induce DNA damage. These effects are important in assessing the role of vitamin C, in the presence of mineral supplements or radioprotective therapeutic agents, particularly in patients with abnormally high tissue copper levels.

Arsenic induces and enhances rat hepatic metallothionein production in vivo.

Metallothionein genes (MT) are inducible by a variety of agents, including heavy metals. We report the induction of MT expression by arsenite (As3+) in rat liver in vivo. As3+ (but not arsenate [As5+]) injection increased MT protein and MT-1 and MT-2 mRNA accumulation in liver only, but not in kidney or pancreas. In addition, As3+ enhanced zinc-induced MT protein accumulation in liver without any increase in MT mRNA levels. These data indicate that arsenic may increase MT expression either directly (by inducing MT mRNA accumulation), or indirectly by altering post-transcriptional events. This constitutes an unusual mechanism of enhancement of MT gene expression and appears to be mediated by processes not specifically associated with binding of arsenite to MT in vivo.

The mutagenicity and cytotoxicity of selenite, "activated" selenite and selenate for normal and DNA repair-deficient human fibroblasts.

At doses varying from 8 x 10(-5) to 3 x 10(-3) M sodium selenite (Na2SeO3) induced DNA fragmentation, DNA-repair synthesis, chromosome aberrations and a mitotic inhibition in cultured human fibroblasts. The response of DNA repair-deficient xeroderma pigmentosum (XP) fibroblasts to selenite is comparable to that of control cells. Incubation with mouse liver S-9 microsomal fraction increased the capacity of selenite to induce chromosome aberrations, DNA-repair synthesis and a lethal effect. XP cells behaved as control cells when treated with activated selenite. Sodium selenate (Na2SeO4) at doses ranging from 8 x 10(-5) to 3 x 10(-3) M could not be activated by incubating with a S-9 preparation. Selenate had the capacity to induce a small but significant DNA-repair synthesis.

Radiation exposure does not alter metallothionein III isoform expression in mouse brain.

The level of metallothionein III mRNA and protein, a brain-specific isoform of metallothionein (MT), was investigated in the brain of MT-I and -II gene knockout (MT-null) mice exposed to 20 Gy whole-body gamma-irradiation. Because MT-null mice did not express MT-I or MT-II isoforms, the total brain MT content in these mice represented the isoform MT-III only. MT-III protein content was determined by a cadmium-binding assay, and the MT mRNA level was measured by reverse transcription followed by polymerase chain reaction (RT-PCR). Both MT-III protein content and mRNA expression in the brains of MT-null mice were not affected by exposure to whole-body irradiation. These results indicate that mouse brain MT-III expression is not induced by ionizing radiation.

Radioimmunoassay of metallothionein in rabbit, rat, mouse, Chinese hamster, and human cells.

We describe a competitive, solid-phase radioimmunoassay for metallothionein, which employs a rabbit antiserum directed against rat MT-2 to detect metallothionein (MT) from several different species (rabbit, mouse, rat, Chinese hamster, and human). The lower limit of detection of the assay for rat MT-2 was 0.7 ng; for rabbit MT-2 it was 2 ng. The method is capable of measuring both isoforms of MT (MT-1 and MT-2). When MT levels in rat and mouse tissues were estimated with this RIA and the silver-saturation method, both assays gave the same pattern of MT induction in control and cadmium-treated animals. Both methods measured high levels of MT in human liver samples. Chinese hamster ovary cells induced with cadmium also showed elevated MT expression. The detectability of MTs from a broad range of species is facilitated by the use of solid-phase MT, which has an avidity for the antiserum similar to that of the MT in the tested sample.

The Effects of N-acetylcysteine on ifosfamide efficacy in a mouse xenograft model.

BACKGROUND/AIM: Nephrotoxicity is observed in 30% of children treated with ifosfamide. We have shown that n-acetylcysteine (NAC) successfully mitigates nephrotoxicity of ifosfamide in cell and rodent models. However, before this treatment is evaluated clinically, it must be established that NAC does not interfere with the efficacy of ifosfamide. MATERIALS AND METHODS: Mice implanted with Ewing's sarcoma tumours received the following treatments: saline, ifosfamide, ifosfamide + NAC concurrently, pre-treatment with NAC + ifosfamide, or NAC alone. RESULTS: Median volumes of EW-7 tumour xenografts in mice treated with ifosfamide (n=8), ifosfamide with concurrent NAC therapy (n=7), and NAC pre-treatment (n=6) (p<0.05) were significantly reduced compared to median tumour volumes of control mice (n=6). None of the NAC treatments affected ifosfamide-mediated reduction in tumour volumes. CONCLUSION: NAC does not interfere with the efficacy of ifosfamide in a EW-7 xenograft model. These results support the clinical evaluation of NAC as a strategy against ifosfamide-induced nephrotoxicity in children.

Antisense treatment in human prostate cancer and melanoma.

Antisense reagents and technology have developed as extraordinarily useful tools for analysis of gene function. The capacity of antisense to reduce expression of RNA (including protein-encoding mRNA and non-coding RNA) important in a multitude of diseases (including cancer) has led to the concept of using antisense molecules as drugs to treat those diseases. Both antisense RNA (RNAi) and antisense oligonucleotides (ASOs) are being developed for this purpose, with ASOs currently the most advanced in clinical testing. ASOs inhibit translation or induce degradation of complementary target RNA, and both Phase I and Phase II trials are either completed or in progress for a number of diseases. In this review, we focus on antisense approaches to treatment of two cancers (melanoma and hormone-resistant prostate cancer) where the early application of ASOs has provided important information revealing both potential for success and lessons for future preclinical and clinical investigation of ASOs as anti-cancer drugs. The progress of clinical application of two ASOs showing promise in treatment of human cancers--Oblimersen (G3139), targeting BCL2 for the treatment of metastatic melanoma, and Custirsen (OGX-11), targeting clusterin for the treatment of hormone refractory prostate cancer (HRPC)--is examined.

Amplification of metallothionein-1 genes in mouse liver cells in situ: extra copies are transcriptionally active.

We have reported previously (J. Koropatnick et al., Nucleic Acids Res 13:5423-5439, 1985) that metallothionein-1 (MT-1) genes in adult mouse liver undergo a two- to three-fold increase in average copy number within 6 hr of treatment of mice with high levels of cadmium salts. The extra copies persist for at least 3 weeks in the absence of subsequent doses of cadmium. We report here that amplified MT-1 genes, which are relatively nuclease resistant early (6 hr) after induction, undergo a change in chromatin structure that renders them nuclease sensitive within 3 days. The change in chromatin structure is accompanied by an increase in the rate of transcription of MT-1 genes to a level approximately twofold higher than that maximally inducible in mouse liver with low MT-1 gene copy number. These data indicate that extra copies of MT-1 genes induced to appear in adult mouse liver cells in situ are, like their counterpart in cultured somatic cells, transcriptionally competent and inducible. However, post-transcriptional events (possibly specific degradation of MT-1 mRNA) have an adverse effect on the level of gene expression at the mRNA level. Two possible mechanisms to explain the appearance of amplified MT-1 genes in organs in situ after heavy metal treatment are discussed. First, de novo amplification of extra MT-1 genes in all, or a subset, of mouse liver cells may be responsible. Alternatively, a portion of mouse liver cells that already possess extra MT-1 genes might be selected for preferential DNA replication.