Nanotechnology-based techniques are being widely evaluated in medical testing and could provide a new generation of diagnostic assays due to their high degrees of sensitivity, high specificity, multiplexing capabilities, and ability to operate without enzymes. In this article, we have modified a nanoparticle-based biobarcode amplification (BCA) assay for early and sensitive detection of HIV-1 capsid (p24) antigen by using antip24 antibody-coated microplates to capture viral antigen (p24) and streptavidin-coated nanoparticle-based biobarcode DNAs for signal amplification, followed by detection using a chip-based scanometric method. The modified BCA assay exhibited a linear dose-dependent pattern within the detection range of 0.1 to 500 pg/ml and was approximately 150-fold more sensitive than conventional enzyme-linked immunosorbent assay (ELISA). No false positive results were observed in 30 HIV-1-negative samples, while all 45 HIV-1 RNA positive samples were found HIV-1 p24 antigen positive by the BCA assay. In addition, the BCA assay detected HIV-1 infection 3 days earlier than ELISA in seroconversion samples. Preliminary evaluation based on testing a small number of samples indicates that the HIV-1 p24 antigen BCA may provide a new tool for sensitive and early detection of HIV-1 p24 antigen in settings where HIV-1 RNA testing is currently not routinely performed.

Regulation of growth, differentiation, and apoptosis by synthetic retinoids can occur through mechanisms that are dependent and independent of their ability to bind and activate nuclear retinoic acid receptors. The objective of this study was to determine if increasing flexibility of the heteroarotinoid structure would affect the specificity of the synthetic retinoids for the receptors and for their regulation of cancerous and nonmalignant cells. Methods were developed to produce the first examples of heteroarotinoids 15a-15h, which contain urea and/or thiourea linking groups between two aryl rings. Substituents at the para position of the single phenyl ring were either an ester, a nitro group, or a sulfonamide group. Ovarian cancer cell lines Caov-3, OVCAR-3, SK-OV-3, UCI-101, and 222 were utilized, and the inhibitory prowess of the heteroarotinoids was referenced to that of 4-HPR (25). Similar to 4-HPR (25), the heteroarotinoids inhibited growth of all cell lines at micromolar concentrations. Although the heteroarotinoids did not activate retinoic acid receptors, the agents induced potent growth inhibition against the cancer cells with weak activity against normal and benign cells. The growth inhibition was associated with cell loss and induction of reactive oxygen species.

Activation of the transcription factor AP-1 (activator protein-1) is required for tumor promotion and maintenance of malignant phenotype. A number of AP-1-regulated genes that play a role in tumor progression have been identified. However, AP-1-regulated genes driving tumor induction are yet to be defined. Previous studies have established that expression of a dominant-negative c-Jun (TAM67) inhibits phorbol 12-tetradecanoyl-13-acetate (TPA)-induced AP-1 transactivation as well as transformation in mouse epidermal JB6/P+ cells and tumor promotion in mouse skin carcinogenesis. In this study, we utilized the tumor promotion-sensitive JB6/P+ cells to identify AP-1-regulated TAM67 target genes and to establish causal significance in transformation for one target gene. A 2700 cDNA microarray was queried with RNA from TPA-treated P+ cells with or without TAM67 expression. Under conditions in which TAM expression inhibited TPA-induced transformation, microarray analysis identified a subset of six genes induced by TPA and suppressed by TAM67. One of the identified genes, the high-mobility group protein A1 (Hmga1) is induced by TPA in P+, but not in transformation-resistant P cells. We show that TPA induction of the architectural transcription factor HMGA1 is inhibited by TAM67, is extracellular-signal-regulated kinase (ERK)-activation dependent, and is mediated by AP-1. HMGA1 antisense construct transfected into P+ cells blocked HMGA1 protein expression and inhibited TPA-induced transformation indicating that HMGA1 is required for transformation. HMGA1 is not however sufficient as HMGA1a or HMGA1b overexpression did not confer transformation sensitivity on P- cells. Although HMGA1 expression is ERK dependent, it is not the only ERK-dependent event required for transformation because it does not suffice to rescue ERK-deficient P- cells. Our study shows (a) TAM 67 when it inhibits AP-1 and transformation, targets a relatively small number of genes; (b) HMGA1, a TAM67 target gene, is causally related to transformation and therefore a potentially important target for cancer prevention.

UNLABELLEDAlthough inducible nitric oxide synthase (iNOS) and nitric oxide (NO) are implicated in tumor pathology, their role in the early stages of carcinogenesis is not well defined. Tumor necrosis factor alpha (TNFalpha) induces iNOS and NO production in transformation-sensitive JB6 P+, but not in transformation-resistant JB6 P-, mouse epidermal cells. We tested the hypothesis that iNOS, by generating NO and reactive nitrogen species, mediates tumor promoter-induced transformation. Specific [N-[3-(aminomethyl)benzyl]acetamidine (1400W)] and non-specific (N(omega)-methyl-L-arginine) iNOS inhibitors significantly reduced TNFalpha-induced NO production in P+ cells but both iNOS inhibitors enhanced TNFalpha-induced anchorage-independent transformation, thus ruling out a mediator role and suggesting an inhibitor role for NO. Independent support for an inhibitor role came from the observation that the NO donor [(Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA/NO)] inhibited TNFalpha- and 12-O-tetradecanoylphorbol-13-acetate-induced transformation. DETA/NO treatment also suppressed tumor phenotype in tumorigenic JB6 RT101 (Tx) cells. Higher concentrations of DETA/NO induced apoptosis. The transformation inhibitory effect of lower DETA/NO concentrations may be attributable in part to inhibition by NO of NF-kappaB-dependent but not of AP-1-dependent transcription.IN CONCLUSION(a) induction of iNOS and NO production does not mediate but actually prevents tumor promotion; (b) iNOS inhibitors enhance the transformation response, and therefore appear not to be appropriate as chemoprevention agents; and (c) NO has both chemopreventive and tumoricidal effects, suggesting promise in cancer chemoprevention and therapy.

Generation of reactive oxygen species (ROS) stimulates transcription by activating transcription factors activator protein 1 (AP-1) and nuclear factor κB (NF-κB). The mouse epidermal JB6 cells constitute a model system that has significantly contributed to the understanding of these events. Clonal variants of JB6 cells are differentially responsive to transformation induced by tumor promoters such as phorbol esters (TPA), epidermal growth factor (EGF) and tumor necrosis factor alpha (TNF-α), as well as oxidative stress. TPA and EGF, acting through the MAP kinase pathway, activate AP-1 and subsequently NF-κB proteins and downstream transcription processes that are involved in the transformation response in transformation-sensitive (P+) JB6 cells. The effect of TNF-α is primarily on the NF-κB pathway. ROS and other free radicals can activate AP-1 and NF-κB transcription coordinately. In JB6 cells, both ERK/Fra-1 and NF-κB activity is essential for the transformation response. Inhibition of NF-κB and AP-1 activity abrogates transformation in JB6 cells as well as in transgenic mice and human keratinocytes. A similar effect is seen with antioxidants, which inhibit NF-κB and AP-1 activity as well as transformation in JB6 cells. The JB6 model is therefore valuable for monitoring early events in oxidative stress related signaling leading to carcinogenesis, and for identifying molecular targets for cancer chemoprevention.

The human malaria parasite survives, lives and multiplies in an immunocompetent host. The parasite deploys various strategies for this purpose, i.e. fluidity in the genome, antigenic variation, molecular mimicry, acquisition of host molecules. During its asexual development in humans, the parasite exchanges a large number of molecules with the host. Deitsch et al. [1] have reported that the parasite also has the ability to import host DNA into its nucleus from the infected host cell and can express it into a protein product. In nature, the parasite has an opportunity of such host DNA import into its nucleus during liver stages as well as blood stages in case of Plasmodium vivax, which prefers to infect human reticulocytes rather than mature anucleated erythrocytes. This corroborates our earlier findings where we reported the presence of Alu elements in a P. vivax antigen [2]. The current methods of parasite purification and subsequent DNA extraction can not remove host DNA if it has either transiently been transferred to the parasite or has become part of the parasite genome. Therefore, in both cases these sequences will be detected as part of the parasite DNA. This would have resulted in the reported host sequences (Alu and LINE), deposited by several labs in the databases of the malarial genome projects. However, Deitsch et al. [3] suggest that this could be a contaminating artifact due to erroneous DNA extraction methods. We partly agree with them because sometimes the host nucleated cells are not completely removed during purification of infected erythrocytes from patient's blood. This might lead to host DNA contamination in parasite DNA preparation. But how one would get rid of the host DNA if it is present intrinsically in parasite's nucleus, either free or in integrated form, after it was horizontally transferred in nature. This possibility exists more in the field isolates for P. vivax. In their experiments Deitsch et al. were unable to detect the Alu/LINE host sequences in the P. vivax by polymerase chain reaction (PCR) but obtained a positive signal on pulse field gel electrophoresis (PFGE) [3]. These results are contradictory to each other because one would presume that the parasites were purified from the rest of the host nucleated cells. Then why would the same parasite preparation produce negative results in PCR and positive in PFGE? In all probabilities one would expect the opposite because PCR can detect a lower level of DNA contamination than PFGE. However, caution should be taken while addressing the issue of host DNA contamination based on PCR results. This is because PCR could be inaccurate as any change (mutation) introduced in synthetic (primer) or template (host or parasite) DNA might give rise to erroneous results. Deitsch et al. [3] have used different primer sequences and DNA templates than in reference [2]. In addition, they have used blood from monkey infected with P. vivax rather than human patients’ blood from endemic areas, a different source of control human DNA, and most importantly different primer sequences. They have used four different primers (F10+B7 and F6+B3) based on Plasmodium falciparum data of Cheng et al. [4] as compared to three primers used in P. vivax data [2] where reverse primer 12R was common (12F+12R and AF+12R). Deitsch et al. [3] have used PFGE data to support their PCR results. The supportive data from PFGE are, however, not very convincing. Firstly, there is hybridization signal in high mol wt DNA that should have been enough to give a positive signal on PCR. Secondly, the lower mol wt DNA is run out. Therefore, these PFGE data may not rule out the transient horizontal transfer of host DNA from reticulocytes/hepatocytes to the parasite. Deitsch et al. [3] have also pointed out the presence of these host sequences in the cultivable human malaria parasite P. falciparum, deposited in genome project databases and in a report [4]. While the chances of host DNA contamination are high in case of P. vivax, where the starting material is patient or monkey infected blood, this should be minimum or absent in case of P. falciparum, which is cultured in vitro using human erythrocytes free from the host nucleated cells. Then the question arises, of where do these host DNA sequences originate from, in case of P. falciparum, if they are not present in the parasite? It seems most likely that these host sequences are in the parasite, either in the transient state or in the integrated form. The malaria parasite has excellent survival skills [5, 6]. It can survive various drug treatments as well as the host's defense system [7]. The parasite uses host machinery to its optimum level. It imports a large number of molecules from the host either for its metabolic system or to evade the host immune system [8]. In the latter case, the parasite uses several mechanisms including molecular mimicry, i.e. presence of host sequences in parasite antigens, acquisition of antibody (IgG and IgM) molecules on the surface of knobs of the P. falciparum infected erythrocytes [9, 10]. The presence of auto-antibodies in the malarial patients’ sera also support this view [11]. However, it is possible that not all of the host molecules are imported but some are synthesized by the parasite. This seems to be possible in the light of experiments showing that the malarial parasite can import and express the host DNA [1]. Therefore, the possibility of the presence of host DNA in the parasite exists whether it is transiently acquired through horizontal transfer or is integrated in its genome. However, the clear picture of host DNA in the parasite will emerge after the completion of the parasite genome projects.

Three heteroarotinoids containing a nitrogen atom in the first ring and a C-O linking group between the two aryl rings were synthesized and evaluated for RAR and RXR retinoid receptor transactivation, tumor cell growth inhibition, and transglutaminase (TCase) induction. Ethyl 4-(N,4,4-trimethyl-1,2,3,4-tetrahydroquinolinyl)benzoate (1) contained an N-CH3 group and activated all retinoid receptors except for RAR gamma. Inceasing the hydrophobicity around the rings with analogues ethyl 4-(N,4,4,7-tetramethyl-1,2,3,4-tetrahydroquinolin-6-oyloxy)benzoate (2) [7-methyl group added] and ethyl 4-(4,4-dimethyl-N-isopropyl-1,2,3,4-tetrahydroquinolin-6-oyloxy)benzoate (3) [NCH(CH3)2 group at C-4] increased the potency and specificity for RAR alpha, RAR beta, and RXR alpha, compared to 1, but had little effect on RXR beta and RXR gamma activation. Although 1 and 3 were unable to activate RAR gamma, 2 did activate this receptor with efficacy and high potency equal to that of 9-cis-retinoic acid (9-c-RA). All three heteroarotinoids exhibited 5-8-fold greater specificities for RAR beta over RAR alpha. In addition, esters 1-3 inhibited the growth of two cell lines each derived from cervix, vulvar, ovarian, and head/neck tumors with similar efficiencies to that of 9-c-RA through a mechanism independent of apoptosis. The vulvar cell lines were the most sensitive, and the ovarian lines were the least sensitive. Ester 2 was similar to 1 and 3 except that 2 was a much more potent growth inhibitor of the two vulvar cell lines, which is consistent with strong RAR gamma activation by 2 (but not by 1 and 3) and the high levels of RAR gamma expression in skin. All three heteroarotinoids induced production of TGase, a marker of retinoid activity in human erythroleukemic cells. Esters 2 and 3 were the more potent TGase activators than 1, in agreement with the stronger activation of the RAR receptors by 2 and 3. The biological activities of these agents, and the RAR gamma potency of 2 in particular, demonstrate the promise of these compounds as pharmaceutics for cancer and skin disorders.

A class of less toxic retinoids, called heteroarotinoids, was evaluated for their molecular mechanism of growth inhibition of two head and neck squamous cell carcinoma (HNSCC) cell lines SCC-2 and SCC-38. A series of 14 heteroarotinoids were screened for growth inhibition activity in vitro. The two most active compounds, one that contained an oxygen heteroatom (6) and the other a sulfur heteroatom (16), were evaluated in a xenograph model of tumor establishment in nude mice. Five days after subcutaneous injection of 10(7) SCC-38 cells, groups of 5 nu/nu mice were gavaged daily (5 days/week for 4 weeks) with 20 mg/kg/day of all-trans-retinoic acid (t-RA, 1), 10 mg/kg/day of 6, 10 mg/kg/day of 16, or sesame oil. After a few days, the dose of t-RA (1) was decreased to 10 mg/kg/day to alleviate the side effects of eczema and bone fracture. No significant toxic effects were observed in the heteroarotinoid groups. All three retinoids caused a statistically significant reduction in tumor size as determined by the Student t-test (P < 0. 05). Complete tumor regression was noted in 3 of 5 mice treated with t-RA (1), 4 of 5 mice treated with 16, 1 of 5 mice treated with 6, and 1 of 5 mice treated with sesame oil. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to determine that the expression levels of RARalpha, RXRalpha, and RXRbeta were similar in the two cell lines, while RARbeta expression was higher in SCC-2 over SCC-38, and RARgamma expression was higher in SCC-38 over SCC-2. Receptor cotransfection assays in CV-1 cells demonstrated that 16 was a potent activator of both RAR and RXR receptors, while 6 was selective for the RXR receptors. Transient cotransfection assays in CV-1 cells using an AP-1 responsive reporter plasmid demonstrated that t-RA (1), 6, and 16 each inhibited AP-1-driven transcription in this cell line. In conclusion, the growth inhibition activity of the RXR-selective 6 and the more potent growth inhibition activity of the RAR/RXR pan-agonist 16 implicate both RARs and RXRs in the molecular mechanism of retinoid growth inhibition. Moreover, the chemoprevention activity and the lack of toxicity of heteroarotinoids demonstrate their clinical potential in head and neck cancer chemoprevention.

This paper investigates the presence and functionality of retinoid signaling pathways in human urinary bladder carcinoma and SV40-immortalized uroepithelial cell lines. Only two of eight cell lines were proliferation-inhibited by 10 microM of either all-trans or 13-cis-retinoic acid. Transactivation of the CAT gene under control of a retinoid-responsive element demonstrated functionality of the signaling pathway in both sensitive cell lines and four of six resistant cell lines. Relative RT-PCR analysis of a panel of retinoid-responsive and inducible genes demonstrated changes in expression levels of all the genes in response to-retinoic acid treatment together with numerous aberrations dysregulations. We conclude that retinoid signaling may be a target for inactivation during tumorigenesis by uncoupling gene expression, proliferation and differentiation. Therefore retinoids are more likely to be effective for chemoprevention than for treatment of bladder carcinomas.

Plasmodium vivax is a very common human malaria parasite but it is poorly characterized at the molecular level. Here, we describe the isolation and characterization of an antigen coding gene of P. vivax which contains Alu elements. This gene, called Pv‐Alu, is expressed during the erythrocytic phase of the parasite. The encoded 200 amino acid long polypeptide is highly hydrophobic, contains transmembrane domains, and is rich in leucine (19.4%), serine (15.9%), proline (15.4%) and phenylalanine (15.4%). The 5′‐untranslated region and part of the 3′‐end coding region of Pv‐Alu show significant homology to different Alu families. The presence of Alu elements in the coding region of a parasite antigen gene is significant from a functional and evolutionary viewpoint.