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The advent of serum prostate-specific antigen (PSA) as a biomarker has enabled early detection of prostate cancer and, hence, improved clinical outcome. However, a low PSA is not a guarantee of disease-free status, and an elevated PSA is frequently associated with a negative biopsy. Therefore, our goal is to identify molecular markers that can detect prostate cancer with greater specificity in body fluids such as urine or blood. We used the RT-PCR differential display method to first identify mRNA transcripts differentially expressed in tumor vs. patient-matched nontumor prostate tissue. This analysis led to the identification of 44 mRNA transcripts that were expressed differentially in some but not all tumor specimens examined. To identify mRNA transcripts that are differentially expressed in most tumor specimens, we turned to differential display of pooled tissue samples, a technique we name averaged differential expression (ADE). We performed differential display of mRNA from patient-matched nontumor vs. tumor tissue, each pooled from 10 patients with various Gleason scores. Differentially expressed mRNA transcripts identified by ADE were fewer in number, but were expressed in a greater percentage of tumors (>75%) than those identified by differential display of mRNA from individual patient samples. Differential expression of these mRNA transcripts was also detected by RT-PCR in mRNA isolated from urine and blood samples of prostate cancer patients. Our findings demonstrate the principle that specific cDNA probes of frequently differentially expressed mRNA transcripts identified by ADE can be used for the detection of prostate cancer in urine and blood samples. body fluids | differential display

Lack of molecular targets in estrogen receptor-negative (ER-negative) breast cancer is a major therapeutic hurdle. We studied NF-[kappa]B activation in human breast tumors and in carcinoma cell lines. Activated NF-[kappa]B was detected predominantly in ER-negative vs. ER-positive breast tumors and mostly in ER-negative and ErbB2-positive tumors (86%). These in vivo results demonstrate association of activated NF-[kappa]B with a subgroup of human breast tumors and are consistent with previously reported in vitro observations using similar classes of human breast cancer cell lines. Finding such an association suggested functional and biological significance. Immunofluorescence demonstrated increased nuclear p65, a component of the active NF-[kappa]B complex, in cytokeratin 19 (CK19)-positive epithelial ceils of ER-negative/ErbB2-positive tumor samples. In contrast, nuclear NF-[kappa]B was detected mostly in stroma of ER-negative and ErbB2-negative tumors, suggesting a role of activated NF-[kappa]B in intercellular signaling between epithelial and stromal cells in this type of breast cancers. To elucidate roles of activated NF-[kappa]B, we used an ER-negative and ErbB2-positive human breast tumor cell line (SKBr3). The polypeptide heregulin [beta]1 stimulated, and herceptin, the anti-ErbB2 antibody, inhibited, NF-[kappa]B activation in SKBr3 cells. The NF-[kappa]B essential modulator (NEMO)-binding domain (NBD) peptide, an established selective inhibitor of I[kappa]B-kinase (IKK), blocked heregulin-mediated activation of NF-[kappa]B and cell proliferation, and simultaneously induced apoptosis only in proliferating and not resting cells. These results substantiate the hypothesis that certain breast cancer cells rely on NF-[kappa]B for aberrant cell proliferation and simultaneously avoid apoptosis, thus implicating activated NF-[kappa]B as a therapeutic target for distinctive subclasses of ER-negative breast cancers.

The hypothesis of this article is that late onset Alzheimer's disease (AD) is influenced by the availability in brain of retinoic acid (RA), the final product of the vitamin A (retinoid) metabolic cascade. Genetic, metabolic, and environmental/dietary evidence is cited supporting this hypothesis. Significant genetic linkages to AD are demonstrated for markers close to four of the six RA receptors, RA receptor G at 12q13, retinoid X receptor B at 6p21.3, retinoid X receptor G at 1q21, and RA receptor A at 17q21. Three of the four retinol-binding proteins at 3q23 and 10q23 and the RA-degrading cytochrome P450 enzymes at 10q23 and 2p13 map to AD linkages. Synthesis of the evidence supports retinoid hypofunction and impaired transport as contributing factors. These findings suggest testable experiments to determine whether increasing the availability of retinoid in brain, possibly through pharmacologic targeting of the RA receptors and the cytochrome P450 RA-inactivating enzymes, can prevent or decrease amyloid plaque formation.

Most chemotherapeutic drugs kill cancer cells by indirectly activating checkpoint-mediated apoptosis after creating nonselective damage to DNA or microtubules, which accounts for their toxicity toward normal cells. We seek to target cancer cells by directly activating checkpoint regulators without creating such damage. Here, we show that [beta]-lapachone selectively induces apoptosis in cancer cells without causing the death of nontransformed cells in culture. This unusual selectivity against cancer cells is preceded by activation of S-phase checkpoint and selective induction of E2F1, a regulator of checkpoint-mediated apoptosis. This study suggests direct checkpoint activation as a strategy against cancer.

Cyclin E1 (formerly called cyclin E) and the recently described cyclin E2 belong to the family of E-type cyclins that operate during the [G.sub.1]/S phase progression in mammalian cells. The two E-cyclins share a catalytic partner, cyclin-dependent kinase 2 (CDK2), and activate their associated kinase activities at similar times during cell cycle progression. Despite these similarities, it is unknown whether the two proteins perform distinct functions, or, alternatively, they control S-phase entry of different cell types in a tissue-specific fashion. To start addressing in vivo functions of E-cyclins, we determined the expression pattern of cyclins E1 and E2 during normal mouse development. We found that the two E-cyclins showed very similar patterns of expression; both were expressed within the proliferating compartment during embryo development. Analyses of cells and tissues lacking members of the retinoblastoma (pRB) family of proteins revealed that the expression of both cyclins is controlled in a pRB-dependent, but p107- and p130-independent fashion, likely through the pRB-dependent E2F transcription factors. We also found that cyclins E1 and E2 are expressed at high levels in mouse breast tumors driven by the Myc oncogene. Last, we found that cyclin E2 is overexpressed in [approximately equal to]24% of analyzed human mammary carcinomas. Collectively these findings suggest that the expression of cyclins E1 and E2 is governed by similar molecular circuitry.

HIV infection is accompanied by an early immune dysfunction limiting host control of virus and likely contributing to difficulties in achieving a successful vaccine against HIV. We report here that the HIV Tat protein is strongly immunosuppressive, both immediately after immunization of mice with soluble protein (sTat), and in seroconverting humans, and propose that Tat-induced suppression cripples immune surveillance to HIV infection. We show that macrophages are sensitive to sTat stimulation at concentrations 1,000-fold lower (500 pM) than T cells, and this stimulation is accompanied by the immunosuppressive induction of Fas ligand on the macrophage. T cell proliferative defects induced by sTat in vitro can be completely (at lower concentrations of sTat) or partially (at higher concentrations) reversed by antagonists to Fas/Fas ligand interaction. We further report a method to preserve immunogenicity while inactivating Tat immunosuppression through oxidation, which advances the use of oxidized Tat as a component of an anti-HIV vaccine. These observations define additional methods to study the immunosuppressive functions of sTat that now may be rapidly applied to primary isolates from individuals with differing clinical courses. Our findings have immediate relevance for vaccine development, by describing and supporting a strategy that includes inactivated star in a multicomponent, anti-HIV vaccine.

While conducting a search for cell cycle-regulated genes in human mammary carcinoma cells, we identified HSIX1, a recently discovered member of a new homeobox gene subfamily. HSIX1 expression was absent at the onset of and increased toward the end of S phase. Since its expression pattern is suggestive of a role after S phase, we investigated the effect of HSIX1 in the [G.sub.2] cell cycle checkpoint. Overexpression of HSIX1 in MCF7 cells abrogated the [G.sub.2] cell cycle checkpoint in response to x-ray irradiation. HSIX1 expression was absent or very low in normal mammary tissue, but was high in 44% of primary breast cancers and 90% of metastatic lesions. In addition, HSIX1 was expressed in a variety of cancer cell lines, suggesting an important function in multiple tumor types. These data support the role for homeobox genes in tumorigenesis/tumor progression, possibly through a cell cycle function.

We report that human immunodeficiency virus type 1 (HIV-1) has evolved a self-perpetuating mechanism to actively generate cells permissive for productive and cytopathic infection. Only activated T cells can be productively infected, which leads to their rapid depletion (2 x [10.sup.9]/day in an infected individual). Establishment of productive HIV-1 infection therefore requires continual activations from the large pool of quiescent T cells. Tat protein, which is secreted by infected cells, activated uninfected quiescent T cells in vitro and in vivo. These Tat-activated uninfected cells became highly permissive for productive HIV-1 infection. Activation of primary T cells by Tat protein involved integrin receptors and was associated with activation of mitogen-activated protein kinases, including ERK1 and JNK kinase. Accordingly, these primary T cells progressed from [G.sub.0] to the late [G.sub.1] phase of the cell cycle.

A study has been conducted to show that wild-type p53 is a potent suppressor of HIV-1 activation by Tat protein, a major transactivator released from HIV-infected cells as a biologically active protein. Cell extract assays show that accumulation of Tat suppresses HIV-1 infection by inhibiting the transcription of the p53 protein, which leads to expression of genes coding for cancer.

A differential display strategy helps identify a new gene, termed mob-1, which is expressed in ras-transformed cells and is a downstream target of the Ras signaling pathway. A secretory protein similar to IP-10, a proinflammatory cytokine family member, is encoded by mob-1 whose 417-bp promoter has dual NF-kappaB and AP-1 binding sites. Mob-1 expression is activated by both oncogenic Ras and serum growth factors that stimulate endogenous Ras. Oncogenic mutations in Ha-ras can facilitate the constitutive expression of this gene.

An analysis of the NF-kappa-B and Tat-induced superactivation of the human immunodeficiency virus 1 long terminal repeat (HIV-1 LTR) in Jurkat cells revealed the strong inhibitory influence of pentoxifylline (PTX) and/or the Tat inhibitor Ro 24-7429. HIV-1 LTR-regulated gene expression involves the combined effect of both these drugs, which can have extensive treatment applications.

The long terminal repeat (LTR) sequence in type 1 human immunodeficiency virus (HIV-1) is responsible for the activation of the latent provirus. Therefore compounds that suppress the activity of HIV-1 LTR are of potential therapeutic use. Three such compounds are presented, namely, topotecan, a semisynthetic DNA topoisomerase I inhibitor, beta-lapuchone, a plant-derived compound with various pharmacological effects and curcumin, the major active component of tumeric. Drug tests showed that these compounds affect the virus at the proviral stage.

The abnormal expression of various cyclins and cyclin-dependent protein kinases in human breast cancer cells when compared to normal cells was described. Gene amplification and overexpression of cyclin E were found in one of ten breast cancer cells and altered expression of cyclin Eprotein in all tumor cells studied. There was also the untimely appearances of mitotic cyclins prior to G1 cyclins in synchronized normal vs. tumor cells. Theresults suggest that aberrant cyclin expression may be involved in the process of tumor formation.

The Yi protein complexes are DNA-binding proteins that have previously been detected at the S phase of normal A31 cells and have been implicated in the regulation of thymidine kinase transcription. Monitoring of these Yi proteins during the cell cycle showed the presence of a G1-phase-specific Yi1 and a S-phase-specific Yi2. Characterization of these Yi proteins revealed similarities to a murine retinoblastoma protein. Furthermore, a cdc2-like protein was detected in Yi1. This protein kinase may be involved in the phosproylation of Yi1, resulting in the progression of the cell cycle.

The effect of a kinase inhibitor Go6796 on growth of epidermal growth factor (EGF)-stimulated estrogen receptor negative (ER-) breast cancer cells in vivo and role of nuclear factor kappa B (NF-[Kappa]B) on tumorogenesis have been investigated. This was studied in an animal model by implanting ER-- mouse mammary epithelial tumor cells (CSMLO) in syngeneic A-J mice. (i) Local administration of Go6976 an inhibitor of protein kinases C alpha and beta inhibited growth of tumors and caused extensive necrotic degeneration and regression of the tumors without causing any microscopically detectable damage to the vital organs liver and lung. (ii) Stable expression of dominant-negative mutants of the beta subunit (dnlkk[Beta]) of the inhibitory kappa B (I[Kappa]B) kinase (dnlkk) that selectively blocked activation of NF-[Kappa]B caused loss of tumorigenic potential of CSMLO cells. Stable expression of dnlkk[Beta] also blocked phorbol 12-myristate 13-acetate (PMA)-induced activation of NF-[Kappa]B and overexpression of cyclin D1, concomitantly with the loss or reduced tumorigenic potential of these cells. Thus, results from in vivo and in vitro experiments strongly suggest the involvement of NF-[Kappa]B in ER-- mammary epithelial cell-mediated tumorigenesis. We propose that blocking NF-[Kappa]B activation not only inhibits cell proliferation, but also antagonizes the antiapoptotic role of this transcription factor in ER-- breast cancer cells. Thus, NF-[Kappa]B is a potential target for therapy of EGFR family receptor-overexpressing ER-- breast cancers.

Early detection is an effective means of reducing cancer mortality. Here, we describe a highly sensitive high-throughput screen that can identify panels of markers for the early detection of solid tumor cells disseminated in peripheral blood. The method is a two-step combination of differential display and high-sensitivity cDNA arrays. In a primary screen, differential display identified 170 candidate marker genes differentially expressed between breast tumor cells and normal breast epithelial cells. In a secondary screen, high-sensitivity arrays assessed expression levels of these genes in 48 blood samples, 22 from healthy volunteers and 26 from breast cancer patients. Cluster analysis identified a group of 12 genes that were elevated in the blood of cancer patients. Permutation analysis of individual genes defined five core genes (P [is less than or equal to] 0.05, PERMAX test). As a group, the 12 genes generally distinguished accurately between healthy volunteers and patients with breast cancer. Mean expression levels of the 12 genes were elevated in 77% (10 of 13) untreated invasive cancer patients, whereas cluster analysis correctly classified volunteers and patients (P = 0.0022, Fisher's exact test). Quantitative real-time PCR confirmed array results and indicated that the sensitivity of the assay (1:2 x [10.sup.8] transcripts) was sufficient to detect disseminated solid tumor cells in blood. Expression-based blood assays developed with the screening approach described here have the potential to detect and classify solid tumor cells originating from virtually any primary site in the body. breast cancer | hematogeneous dissemination of tumor cells | cDNA | prognostic markers | differential display

The epidermal growth factor (EGF) family of receptors (EGFR) is overproduced in estrogen receptor (ER) negative (-) breast cancer cells. An inverse correlation of the level of EGFR and ER is observed between ER- and ER positive (+) breast cancer cells. A comparative study with EGFR-overproducing ER- and low-level producing ER+ breast cancer cells suggests that EGF is a major growth-stimulating factor for ER- cells. An outline of the pathway for the EGF-induced enhanced proliferation of ER- human breast cancer cells is proposed. The transmission of mitogenic signal induced by EGF-EGFR interaction is mediated via activation of nuclear factor [Kappa]B (NF-[Kappa]B). The basal level of active NF-[Kappa]B in ER- cells is elevated by EGF and inhibited by anti-EGFR antibody (EGFR-Ab), thus qualifying EGF as a NF-[Kappa]B activation factor. NF-[Kappa]B transactivates the cell-cycle regulatory protein, cyclin D1, which causes increased phosphorylation of retinoblastoma protein, more strongly in ER- cells. An inhibitor of phosphatidylinositol 3 kinase, Ly294-002, blocked this event, suggesting a role of the former in the activation of NF-[Kappa]B by EGF. Go6976, a well-characterized NF-[Kappa]B inhibitor, blocked EGF-induced NF-[Kappa]B activation and up-regulation of cell-cycle regulatory proteins. This low molecular weight compound also caused apoptotic death, predominantly more in ER- cells. Thus Go6976 and similar NF-[Kappa]B inhibitors are potentially novel low molecular weight therapeutic agents for treatment of ER- breast cancer patients.

Ablation of tumor colonies was seen in a wide spectrum of human carcinoma cells in culture after treatment with the combination of [Beta]-lapachone and taxol, two low molecular mass compounds. They synergistically induced death of cultured ovarian, breast, prostate, melanoma, lung, colon, and pancreatic cancer cells. This synergism is schedule dependent; namely, taxol must be added either simultaneously or after [Beta]-lapachone. This combination therapy has unusually potent antitumor activity against human ovarian and prostate tumor prexenografted in mice. There is little host toxicity. Cells can commit to apoptosis at cell-cycle checkpoints, a mechanism that eliminates defective cells to ensure the integrity of the genome. We hypothesize that when cells are treated simultaneously with drugs activating more than one different cell-cycle checkpoint, the production of conflicting regulatory signaling molecules induces apoptosis in cancer cells. [Beta]-Lapachone causes cell-cycle delays in late [G.sub.1] and S phase, and taxol arrests cells at [G.sub.2]/M. Cells treated with both drugs were delayed at multiple checkpoints before committing to apoptosis. Our findings suggest an avenue for developing anticancer therapy by exploiting apoptosis-prone 'collisions' at cell-cycle checkpoints.