James Koropatnick

The hyaluronan receptors CD44 and Rhamm (CD168) form complexes with ERK1,2 that sustain high basal motility in breast cancer cells.

CD44 is an integral hyaluronan receptor that can promote or inhibit motogenic signaling in tumor cells. Rhamm is a nonintegral cell surface hyaluronan receptor (CD168) and intracellular protein that promotes cell motility in culture. Here we describe an autocrine mechanism utilizing cell surface Rhamm-CD44 interactions to sustain rapid basal motility in invasive breast cancer cell lines that requires endogenous hyaluronan synthesis and the formation of Rhamm-CD44-ERK1,2 complexes. Motile/invasive MDA-MB-231 and Ras-MCF10A cells produce more endogenous hyaluronan, cell surface CD44 and Rhamm, an oncogenic Rhamm isoform, and exhibit more elevated basal activation of ERK1,2 than less invasive MCF7 and MCF10A breast cancer cells. Furthermore, CD44, Rhamm, and ERK1,2 uniquely co-immunoprecipitate and co-localize in MDA-MB-231 and Ras-MCF10A cells. Combinations of anti-CD44, anti-Rhamm antibodies, and a MEK1 inhibitor (PD098059) had less-than-additive blocking effects, suggesting the action of all three proteins on a common motogenic signaling pathway. Collectively, these results show that cell surface Rhamm and CD44 act together in a hyaluronan-dependent autocrine mechanism to coordinate sustained signaling through ERK1,2, leading to high basal motility of invasive breast cancer cells. Therefore, an effect of CD44 on tumor cell motility may depend in part on its ability to partner with additional proteins, such as cell surface Rhamm.

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.

NON-COVALENTLY FUNCTIONALIZED SINGLE-WALLED CARBON NANOTUBE FOR TOPICAL SIRNA DELIVERY INTO MELANOMA

RNAi can specifically regulate gene expression, but efficient delivery of siRNA in vivo is difficult while it has been shown that modified carbon nanotubes (CNT) protect siRNA, facilitate entry into cells and enhance transdermal drugs delivery. Single-walled carbon nanotubes (SWCNT) were functionalized non-covalently with succinated polyethyleimine (PEI-SA). In this study, the water soluble CNT, PEI-SA/CNT (IS/C) were isolated and characterized, the gene silencing induced by IS/C/siRNA complexes was achieved in vitro in B16-F10 cells. In vivo delivery was topically applied to shaved mouse skin, as well as topically to a C57BL/6 mice melanoma model. We found significant uptake of Cy3-labeled siRNA specific to Braf (siBraf) and gene silencing in the tumor tissue. Treatment with IS/C/siBraf resulted in attenuation of tumor growth over a 25-day period. This new delivery method has provided a new possibility for future siRNA delivery and therapy, which providing insight for the potential application and development of CNT-based siRNA delivery.

Reinstalling Antitumor Immunity by Inhibiting Tumor-Derived Immunosuppressive Molecule IDO through RNA Interference

Tumor-derived immune suppression is a major impediment to successful immune/gene cancer therapy. In the present study, we describe a novel strategy to disrupt tumor-derived immune suppression by silencing a tolerogenic molecule of tumor origin, IDO, using small interfering RNA (siRNA). Silencing of IDO in B16F10 cells in vitro using IDO-siRNA prevented catabolism of tryptophan and inhibited apoptosis of T cells. IDO-siRNA treatment of B16F10 cells in vitro inhibited subsequent growth, tumor formation, and the size of tumor formed, by those cells when transplanted into host mice. In vivo treatment of B16F10 tumor-bearing mice successfully postponed tumor formation time and significantly decreased tumor size. Furthermore, in vivo IDO-siRNA treatment resulted in recovery of T cells responses and enhancement of tumor-specific killing. Thus, silencing IDO may break tumor-derived immune suppression. These data indicate that RNA interference has potential to enhance cancer therapy by reinstalling anticancer immunity.

Detection of circulating tumor cells in advanced head and neck cancer using the cellsearch system

Early detection of circulating tumor cells (CTCs) offers the possibility of improved outcome for patients with head and neck squamous cell cancer (HNSCC).

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

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. 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 μm, ZnCl2 20–40 μm or HgCl2 2 μm 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‐1β (IL‐1β) mRNA accumulation. 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.

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.

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.

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

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‐theonyl‐L‐glutamic acid). 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 μg 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. These data indicate that antisense ODN down‐regulation of TS can inhibit human tumour cell proliferation and enhance the efficacy of TS‐targeted drugs.

A sensitive time-resolved fluorescent immunoassay for metallothionein protein.

Metallothioneins (MTs) are a family of low molecular weight metal-binding proteins induced by a broad range of stress conditions, including exposure to transition metal ions. Biochemical and immunological methods to measure MT protein levels in tissues and cultured cells have been reported, but accuracy and sensitivity is impeded by high background levels, low specificity of currently available reagents, and relatively laborious and time-consuming multistep procedures. To address these difficulties, a protocol has been developed to measure MT protein levels using a competitive solid phase assay based on dissociation enhanced lanthanide fluoroimmuno (DELFIA) detection of anti-MT monoclonal antibody bound to solid phase MT. This assay allows time-resolved detection of antibody binding, based on binding and exchange of different lanthanide chelates followed by fluorescent detection, designed to reduce background fluorescence and increase sensitivity. The method allows measurement of low MT levels that are undetectable using current radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) protocols, and yields reproducible results with low background over a wide range of MT concentrations. Improved sensitivity of MT protein detection is of value in toxicological measurement of stress responses and assessment of MT expression and function.