Depression in Patients With Intracranial Hemorrhage Secondary to Traumatic Brain Injury.

This article discusses the prevalence of depression in patients with intracranial hemorrhage (ICH) and the relationship of selective serotonin reuptake inhibitor (SSRI) use with bleeding risk. A detailed account of the patient's psychiatric history and current hospital admission is also provided. This article then further explores the pathophysiological mechanisms that contribute to depression in ICH patients, the effect of SSRIs on outcomes in patients with ICH, and ways to treat depression in ICH patients. Based on the literature, the conclusion is that practitioners should avoid SSRIs in ICH patients with certain genetic markers and treat depression as seriously as one would treat a physical ailment. Ultimately, more research is necessary to explore how to treat depression in this patient population.

Normoxic or hypoxic CD44/CD41 a2 B1 integrin-positive prostate PC3 cell side fractions and cancer stem cells.

A CD44/a 2 B 1- (CD41 integrin) and B-catenin-labeled fraction of PC3 prostate cancer cells is able to reconstitute cells representative of the original tumor in immuno-deficient mice (Li et al. in Cancer Res 68:1820-1825, 2008). After 48 h of culture under nitrogen with a resulting medium pH of 7.8, sorted hypoxic PC3 cells yielded a higher percentage and concentration/10(5) of cells in a doubly labeled (DL) CD44(+)/CD41(+) side fraction compared with control cells cultured under normoxia (5 % CO2 in the ambient atmosphere at 37 °C). When the rise in pH was prevented (95 % N2, 5 % CO2), the difference in sorted hypoxic cell numbers remained. Sorted N and H DL cells and CD44(+)/CD41(-) cells were cultured under standard conditions. After 1-2 weeks, the number of attached colonies from formerly hypoxic cells, whether previously cultured with N2 or 95 % N2 + 5 % CO2, exceeded the number of doubly labeled normoxic cells, consistent with their greater initial concentration. Cultured sorted N or H CD44(+)/CD41(-) cells resulted in monolayers containing a small percentage of DL cells. Recovery of greater percentage and numbers of putative cancer stem cells, confirmed by quantitative cell sorting after culture under hypoxic conditions, is consistent with their greater relative numbers present in hypoxic niches. In addition, the report that neither CD44(+) nor CD41(+) epitopes were preferentially associated with FAM65B(high)/MF12(low)/LEF1(low) PC3 cells able to reconstitute tumors in immuno-deficient mice (Zhang and Waxman in Mol Cancer 9:319-330, 2010) suggests an in vitro mimic of tumor cell heterogeneity observed in epithelial cancers.

The effect of normoxia and hypoxia on a prostate (PC-3) CD44/CD41 cell side fraction.

It has been reported that human prostate-derived PC-3 cells that are CD44- and CD 41 (a2 B1)-positive are enriched in cancer stem cells. This study compared the effect of PC-3 cell proliferation under normoxia or hypoxia on the initial and subsequent expression of this doubly-labeled side-fraction. Despite the numerical advantage of attached normoxic cells, 48 h of culture under nitrogen, an environment containing minimal oxygen and CO(2) resulting in an elevated pH of the medium, was associated with a higher percentage, absolute and relative number of doubly-labeled (DL) hypoxic compared to normoxic cells. At 24 h, the reverse was found. When the pH was controlled with the use of 95% nitrogen and 5% carbon dioxide, the percentage and number of normoxic DL cells exceeded hypoxic ones at both 24 and 48 h. At 24 h, 2-deoxy-L-glucose or sodium arsenate reduced normoxic DL cell numbers more than hypoxic ones. The interplay between hypoxia, increased medium pH and the effect of inhibitors as they might influence therapy are considered.

Are cancer stem cells concentrated in more alkaline hypoxic regions of tumors?

We wonder if the most viable hypoxic cancer stem cells concentrate in more alkaline regions of tumors, favoring their survival and evolution. Alternately, or in addition, do some cancer stem cells themselves maintain a more alkaline internal environment, achieving the same result. Based upon the response of cultured cells, including stem cells, to a certain degree of hypoxia and of most if not all proliferating cells to a somewhat more alkaline ambient and especially endogenous pH, their survival and proliferation should be favored. The broad outline of the argument, abstracted from a number of the available examples is developed: that the survival of cancer stem cells is favored by these conditions, contributing to their limited response to various therapies and their subsequent development of more malignant properties.

In vitro effects of dichloroacetate and CO2 on hypoxic HeLa cells.

HeLa and PANC-1 cells were exposed to conflicting signals promoting anaerobic or aerobic energy-generating processes and their viability, cell numbers and the ability of HeLa cells to form colonies were assessed. Under conventional aerobic cell culture with 5% CO(2), dichloroacetate (DCA), an inhibitor of the enzyme pyruvate dehydrogense kinase with subsequent stimulation of pyruvate dehydrogenase that redirects energy metabolism toward the Kreb cycle, reduced HeLa and PANC-1 cellular proliferation and viability. With nitrogen-induced hypoxia, the number of control cells and cells cultured with 12.5 mM DCA paradoxically was greater than that of normoxic controls under similar conditions. A higher medium pH of cells cultured under nitrogen contributed to these differences. In 96-well experiments, 95% nitrogen with 5% CO(2) reduced the numbers of hypoxic cells and medium pH toward that of the aerobic controls, with retention of the DCA-induced hypoxic compared to normoxic cell numbers. The media of these cells cultured with DCA still exhibited an increased pH. Increased hypoxia-inducible factor 1, alpha subunit (HIF1A) mRNA expression in hypoxic HeLa cells and their greater reliance on D-glucose for metabolic energy confirmed the reliability of the incubation conditions. Compared with normoxic cells, hypoxic cells initially increased their synthesis of ATP, but once proliferation ceased, this no longer closely correlated with cell numbers. Type 1 apoptosis, which was somewhat greater in hypoxic than normoxic cells, contributed to hypoxia and DCA-induced cell death. Colony counts of hypoxic, DCA-inhibited cells subsequently switched to normoxia exceeded those of similarly treated normoxic DCA cells. Despite inhibition in certain hypoxic environments of pyruvate dehydrogenase kinase by DCA and its contribution to increased cellular apoptosis and necrosis, hypoxic cells generally outnumbered normoxic control cells, as did hypoxic DCA-treated cells compared with comparable DCA-treated normoxic cells. Since in vivo hypoxic cells are considered a major factor contributing to therapeutic failure, and as DCA redirects energy metabolism toward the more energy efficient Kreb citric acid cycle, associated with increased medium (and inferred cellular) pH, similar circumstances in vivo could promote proliferation and survival of hypoxic cell clones with the potential for developing unwanted properties.

Do intra-tumor alkaline micro-regions represent additional therapeutically privileged sites?

We briefly review some implications for therapeutic resistance in solid cancers that could be associated with more alkaline intra-tumor micro-regions reported to exist. Regions of increased alkalinity may provide a proliferative advantage for cancer "stem" or other cells, as more alkaline intra- and extra-cellular environments often are associated with increased cellular proliferation. If increased alkalinity is present in aerobic, but perhaps more especially in hypoxic micro-environments, proliferation of cells less susceptible to programmed cell death, with reduced expression of multi-drug resistance membrane proteins and altered efficacy of some therapeutic drugs should develop. Such cells are also more likely to generate aberrant clones resistant to additional therapy, particularly those dependent upon mitochondrial oxidative processes with greater generation of free radicals, compared to cells reliant on anaerobic glycolysis for metabolic energy. The interplay between alkalinity and normoxia, hypoxia or anoxia may differentially advantage some cancer "stem" cells.

Does homeobox-related "positional" genomic information contribute to implantation of metastatic cancer cells at non-random sites?

Reasons for the lodgment of metastases from several types of solid cancer at apparently non-random sites have not been established. Recently, a group of genes expressed in human fibroblasts obtained from different anatomic locations was implicated in "positional" genomic information. Essentially, a Cartesian coordinate system identifying fibroblasts originally resident at anterior or more posterior, proximal or distal and dermal or non-dermal (heart, lung, etc.) locations was proposed. The determinants used for these identifications included HOX genes, central to embryonic segmental development, some of which are expressed in differentiated, post-embryonic cells. To the extent that HOX or other homeobox genes are expressed in ectodermal, mesodermal or endodermally-derived, malignantly transformed cells, they might contribute "positional" information to nidation of specific malignant clones at non-random sites. As understood in the past, interdiction of HOX or homeobox-related gene expression might reduce the probability of cancer cell implantation or alter their destinations in complex ways. Ideally, by interfering with HOX or other homeobox gene-related expression of antigenic determinants potentially contributing to their "homing" and nidation, reduced implantation of circulating cancer cells could render them more susceptible to systemic chemotherapy or immunotherapy, as demonstrated in mice. Furthermore, HOX or other homeobox genes or their products could provide novel intra- or extracellular targets for therapy.

Outcome of a national Israeli cohort of pediatric systemic lupus erythematosus.

The aim of this study was to describe the clinical manifestations and outcomes of a national cohort of childhood systemic lupus erythematosus (cSLE). All cases of cSLE registered in the Israeli national registry of children with rheumatic diseases between 1987-2003 were examined for disease activity and damage by the SLE disease activity index (SLEDAI) and SLE collaborating clinics/American College of Rheumatology (SLICC/ACR) damage index. Demographic, clinical, laboratory and treatment factors were analysed for their effect on the outcome. One-hundred and two patients were identified, 81% females, with a mean age at diagnosis of 13.3 +/- 2.6 years. The mean SLEDAI score was 17.2 +/- 9.0 (range 2-60). Fifty four patients were followed for at least five years. The mean SLEDAI decreased to 7.6 +/- 6.3 (0-29) and the mean SLICC/ACR damage index was 0.7 +/- 1.6 (0-8). Five patients developed chronic renal failure. No patients died. No factors were found to be significantly associated with the outcome except the initial SLEDAI score. The five-year outcome of our national cSLE cohort was good; with relatively low activity and minimal damage in most patients. The initial SLEDAI predicted the development of late damage.

To what extent are the "sets" of biologic properties expressed by hypoxic cancer cells and by cancer "stem" cells equivalent, intersecting, disjoint or complementary? Some implications for the design of cancer therapies.

Resistance to various cancer therapies with survival or recurrence of a malignancy has been ascribed to the inability to "kill" hypoxic cancer cells. The reported resistance of cancer "stem" cells has also been proposed as a major reason for this outcome. In planning therapy, it should be important to know whether these two categories "overlap": do "hypoxic" cells subsume cancer "stem" cells; alternately, are "stem" cells somewhat hypoxic or are these categories distinct? If the former is true and these categories overlap, to what extent do their properties share biochemical elements in common; if the latter is the case, should both properties be "targeted" independently? The inability of a proposed therapy to suppress these foci of resistance could preclude a successful outcome. Results from pre-clinical and clinical laboratory determination of the stem cell/hypoxic cell response may reflect the likely outcome of the proposed clinical treatment.

Bispecific antisense oligonucleotides with multiple binding sites for the treatment of prostate tumors and their applicability to combination therapy.

Antisense oligonucleotides (oligos) against transforming growth factor-alpha (TGF-alpha; MR(1)) and its binding site, the epidermal growth factor receptor (EGFR; MR(2)), have proven efficacious against PC-3 and LNCaP prostate tumors when evaluated in both in vitro and in vivo models. To enhance their activity, and also to introduce a significantly different type of multifunctional agent into this field, "bispecific" oligos were constructed containing truncated sequences (derived from MR(1) and MR(2)) recognizing both TGF-alpha and EGFR mRNA internal binding sites, located about their respective AUG initiation codons. Two bispecifics were constructed, each having complementary sequences for TGF-alpha and EGFR mRNA, but differing in their 5' to 3' tandem orientation (TGF-alpha/EGFR [MR(12)] and EGFR/TGF-alpha [MR(21)] sequences). These bispecifics were tested in an in vitro system against PC-3 and LNCaP prostate tumor cells, with comparisons made to the original monospecific oligos from which they were derived. Efficacy was also compared when administered either alone or in combination with conventional chemotherapeutic agents. The purpose of this study was: 1) to validate the concept that these newly developed bispecific oligos have antitumor activity; 2) to enhance their efficacy through combination therapy; 3) to identify differences in effectiveness dependent upon binding site orientation; 4) identification of a dominant binding site that can be used to design other bispecifics that target additional tumor regulatory pathways. When fully evaluated against PC-3 cells in a series of experiments, newly developed bispecific oligos are at least as effective as their monospecific counterparts from which they were derived, and the bispecific with the MR(21) orientation is notably more effective than the MR(1) monospecific by 64% (p = 0.014 by Student t-test and p = 0.068 by the more stringent Mann-Whitney U test). Bispecifics were more effective when administered with chemotherapeutics (producing inhibition of 52.1% and 61.2% for MR(12) and MR(21), respectively, with Cytoxan (cyclophosphamide) inhibition of 59.0% and 65.1% for MR(12) and MR(21), respectively, with Taxol (paclitaxel) and 63.0% and 69.4% for MR(12) and MR(21), respectively, with DES [diethylstilbestrol]). Increasing the oligo concentration above 6.25 microM with cyclophosphamide had no additional effect. The sequence directed against EGFR was dominant and contributed most to bispecific activity, particularly when inserted 5' to the TGF-alpha binding sequence (MR(21) orientation). Bispecific oligos are a significant advance in the design of antisense compounds and could play a role in treating prostate cancer, particularly when they are administered with traditional chemotherapeutics. The truncated portion of the MR(2) oligo used here should be included when constructing second-generation bispecifics that target proteins associated with other regulatory pathways, such as apoptosis.

The proliferative response of hela cells to 2-deoxy-D-glucose under hypoxic or anoxic conditions: an analogue for studying some properties of in vivo solid cancers.

BACKGROUND: Hypoxic cancer cells located beyond the diffusion path of sufficient oxygen are considered a nidus of therapeutic failure. Due to the dependence of many malignantly transformed cells on glycolysis for metabolic energy, inhibiting this and other sources of energy should seriously reduce cell viability and proliferation, additively or even synergistically. MATERIALS AND METHODS: To try and duplicate in vitro some of the features of in vivo cancer cells likely to resist therapy, HeLa cells were incubated with sub-lethal concentrations of 2-deoxy-D-glucose under aerobic, hypoxic or virtually anoxic conditions, verified by increased synthesis of Hif-1alpha, and their replication and survival determined. MK 886, an inhibitor of mitochondrial function was used to estimate participation of that organelle in energy metabolism. RESULTS: Culture of cervical cancer-derived HeLa cells with 2-deoxy-D-glucose under these restrictive conditions did not reduce their proliferation or viability to the expected extent. Their surprisingly robust survival included the anticipated increase in dependence upon glycolysis and implied a likely entrainment of other constitutive and possibly facultative energy sources and pathways. Increased synthesis of Hif-1alpha, increased binding to its consensus sequence and reduced inhibition from MK 886 in cells under oxygen-deficient environments confirmed the presence of restrictive conditions. CONCLUSION: Efforts to suppress HeLa cell survival by reducing glucose consumption and metabolic energy from ambient oxygen may require inhibition of multiple energy sources, possibly not all of them identified. In vitro assessment of agents directed against sources of metabolic energy or of other therapeutic agents against these or additional potential targets should include studies under hypoxia and relative anoxia. In this way, the possible responses of in vivo hypoxic or anoxic cancer cells believed to contribute to therapeutic failure may be estimated.

Reactive oxygen species and redox-induced programmed cell death due to MK 886: cells ("soil") "trump" agent ("seed").

Micromolar concentrations of the five-lipoxygenase inhibitor, MK 886 induce a "type 1" (apoptotic, extrinsic, death domain, receptor-dependent, caspase-positive) form of programmed cell death in Bcl-2-positive U937 human monoblastoid and HL-60 myeloid leukemia cells. A "type 2" (intrinsic, mitochondria-dependent, autophagic, in some examples caspase-negative (Panc-1)) form is induced in Panc-1 pancreatic and PC3 prostate cell lines. The latter two lines from epithelial-derived solid human cancers are Bcl-2-negative. Micromolar MK 886 induces an acute rise in Ca2+ in washed, Ca2+-poor U937 and HL60 cells in Ca2+ and Mg2+-free Hank's buffer. In U937 cells, much of the increase, or more properly redistribution, is nuclear in location (HL-60 not tested). No MK-886-induced acute Ca2+ increase developed in Panc-1 or PC3 cells. Bcl-2-positive HeLa cervical cancer cells exhibited an acute MK 886-induced increase in Ca2+. In the U937, PC3 and Panc-1 cells examined, MK-886 rapidly increased oxidative stress and decreased mitochondrial membrane potential, indicating that neither event is directly determinative for the altered distribution of Ca2+ or the form of PCD observed. Inhibition of increased U937 Ca2+ by the anti-oxidant, N-acetyl-L-cysteine, the effects of inhibitors of mitochondrial function including antimycin A, atractyloside, cyclosporin A, the L/N channel blocker loperamide, the intracellular chelator BAPTA and 2 agents, HA-14 and 3-methyl-antimycin A3 that impair Bcl-2 function further define these events. These differences in the Ca2+ response and possibly also the form of PCD that results may depend upon the presence of Bcl-2 or a related protein participating in a juxta-nuclear / nuclear Ca2+ ion channel. The role of mitochondria, the mechanism by which increased oxidative stress initiates the rapid release of Ca2+ from intracellular, possibly juxta-nuclear / nuclear sites or its redistribution to U937 Ca2+ nuclei, and whether this "signal" or possibly even ROS themselves mandate the type of PCD observed, presumably by differential modulation of transcription, remain to be determined. Lastly, these results demonstrate that, as might be expected, "soil" (cell type) trumps "seed" (inciting agent)".

Disparate forms of MK 886-induced programmed death in BCL-2 (+) blood and BCL-2 (-) solid cancer cells and a putative "nuclear" Ca2+ channel: "soil" trumps "seed"?

UNLABELLED: The five-lipoxygenase inhibitor, MK 886, in micromolar concentration induces a "type 1" form of programmed cell death in U937 human monoblastoid cells and a "type 2" form in Panc-1 pancreatic and PC3 prostate cell lines. The latter two lines originate from epithelial-derived solid human cancers. An acute rise in Ca(2+) occurs in U937 and HL 60 myeloid cells, in U937 cells located in their nuclei (HL 60 not tested), both of which are Bcl-2 positive. The two solid cancer cell lines express neither of these features. Solid tumor-derived Bcl-2-positive HeLa cervical cancer cells exhibit an acute increase in Ca(2+) after challenge with MK 886. In U937, PC3 and Panc-1 cells tested, the agent acutely increases oxidative stress and decreases mitochondrial membrane potential, indicating that neither event is directly determinative for the form of PCD. The role of mitochondria and the mechanism by which increased oxidative stress initiates the acute rise in U937 "nuclear" Ca(2+), the contribution, if any, of Bcl-2 in initiating the Ca(2+) signal and the latter in mandating the type of PCD, presumably through differential modulation of transcription, remain to be determined. Lastly, these results demonstrate that "soil" trumps "seed". HYPOTHESIS: Despite similarities in response, including those of the mitochondria to micromolar concentrations of MK 886, hematopoietic and epithelial-derived non-hematopoietic solid cancer cell lines exhibit dissimilar forms of programmed cell death. These differences may depend upon the presence of Bcl-2 or a related protein participating in a juxta-nuclear/nuclear Ca(2+) ion-channel. Evidence for this supposition is discussed.

Acute changes in U937 nuclear Ca2+ preceding type 1 "apoptotic" programmed cell death due to MK 886.

BACKGROUND: MK 886, a 5-lipoxygenase inhibitor, induces a type 1 "apoptotic" form of programmed cell death in Bcl-2-positive U937 monoblastoid cells. In Ca2+-depleted, nonpermeabilized U937 cells studied with MK 886 in a Ca2+-free medium, an acute increase in Ca2+ occured within 10 to 20 seconds, detected with fura-2 measured with a spectrofluorimeter. METHODS AND RESULTS: The increased fluorescence was nuclear in location, as judged by confocal microscopy. The antioxidant, N-acetyl-L-cysteine, three agents that inhibit mitochondrialfunction at identified sites, antimycin A, atractyloside and cyclosporin A, the L/N-channel inhibitor, loperamide and BAPTA, an intracellular Ca+ chelator preloaded into cells each reduced the extent or prevented the acute MK 886-induced rise in Ca2+, as determined by radiometric detection. Rhodamine-2, a more selective mitochondrial Ca2+ probe, provided no evidence for nuclear Ca2+ originating from that extra-nuclear site or from the endoplasmic reticulum. With 2', 7'-dichloro-dihydrofluorescein-labelled cells to detect reactive oxygen species, MK 886 increased the initial fluorescent signal from a number of intracellular, largely extra-nuclear sites, including mitochondria. Two chemicals that inhibit the function of Bcl-2, HA14-1 and 2-methyl-antimycin A3, reduced the Ca2+ response to MK 886, if pre-incubated with the Bcl-2-positive U937 cells at 37 degrees C for several hours. MK 886 was previously shown to induce reactive oxygen species and a fall in mitochondrial membrane potential in both Bcl-2-positive U937 and in Bcl-2-negative PC-3 prostate and panc-1 pancreatic cancer cells. The latter solid tumor cells undergo an atypical "type 2" PCD without an acute rise in nuclear Ca2+. CONCLUSION: These results are consistent with an MK 886-induced increase of reactive oxygen species from intra-cellular sites including mitochondria which release Ca2+ located primarily at or near nuclei. These events may involve Bcl-2 participating in some form of Ca2+ channel and nuclear Ca2+ binding proteins undergoing conformational changes due to reactive oxygen species. Reasons for the different PCD responses in Bcl-2 positive lympho-hematopoietic compared to Bcl-2-negative solid cancer cell lines, respectively with and without the induced nuclear Ca2+ signal, remain to be defined.

Backbone modification alters the efficacy of antisense oligonucleotides directed against mRNA encoding either TGF-alpha or EGFR in the treatment of prostate cancer cell lines.

Antisense oligonucleotides (oligos) directed against mRNA-encoding, transforming growth factor-alpha (TGF-alpha) and the epidermal growth factor receptor (EGFR), have been shown to significantly inhibit in vitro and in vivo growth of prostate tumor models. Recently, second generation oligos have been employed with identical base sequences, but containing backbome modifications that enhance stability, solubility and circulatory patterns. Using relatively low concentrations of oligos, we compared the efficacy of the first generation phosphorothioated oligos against TGF-alpha (MR1) and EGFR (MR2) with second generation oligos containing completely phosphorothioated backbones and different patterns of 2'-methoxyethyl (2'-MOE) backbone modifications, while retaining the original designated base sequence using, the LNCaP and PC-3 prostate cancer cell lines, respectively. All experiments were conducted in vitro with lipofectin to enhance oligo entry. Under these conditions, using oligo concentrations between 0.83 and 3.32 microM for LNCaP cells treated with oligos directed against TGF-alpha only the first generation MR1 had inhibitory activity. When treated with oligos directed against EGFR, none of the oligos had inhibitory activity and they behaved similarly. Using the PC-3 cell line and treatment directed against TGF-alpha with oligo concentrations between 0.42 and 3.32 microM, first generation MR1 and second generation 5005 behaved similarly with no notable effect, while second generation 5007 produced dramatic growth stimulation. When PC-3 cells were treated with oligos directed against EGFR, second generation 5006 and 5008 had similar and apparently dose-dependent inhibition. We conclude that backbone modifications influence oligo efficacy and may result in either enhanced or diminished activity. Because of their activity against the hormone insensitive PC-3 cells, the 5006 and 5008 compounds warrant additional study at greater concentrations and also merit in vivo testing.

Effectiveness of and adverse events after percutaneous coronary intervention in patients with mild versus severe renal failure.

Patients with renal failure undergoing percutaneous coronary intervention (PCI) experience reduced procedural success rates and increased in-hospital and long-term follow-up major adverse cardiac events. This study was designed to determine whether the severity of preprocedural renal failure influences the outcomes of patients with renal failure undergoing PCI. We compared the immediate and long-term outcomes of 192 patients with mild renal failure (creatinine 1.6 to 2.0 mg/dl, mean 1.76) with those of 131 patients with severe renal failure (creatinine >2.0 mg/dl, mean 2.90), selected from 3,334 consecutive patients undergoing PCI between 1994 and 1997. Although the overall population with renal failure represents a high-risk group, the severe renal failure cohort had a higher incidence of hypertension, multivessel disease, prior coronary bypass surgery, vascular disease, and congestive heart failure (all p values <0.05), yet had similar angiographic characteristics. Procedural success was higher in the group with severe renal failure (93.7% vs 87.7%, p = 0.04). There were no statistically significant differences in in-hospital mortality (11.5% vs 9.9%, p = 0.7), Q-wave myocardial infarction (0.5% vs 0%, p = 0.4), emergent bypass surgery (0% vs 0%, p = 1.0), and in-hospital major adverse cardiac events (11.5% vs 9.9%, p = 0.7) between the mild and severe renal groups, respectively. Kaplan-Meier analyses showed no statistically significant difference in long-term survival (log rank test, p = 0.1) or event-free survival (log rank test, p = 0.3) between the 2 groups. Finally, creatinine was not identified as an independent predictor of in-hospital or long-term follow-up major adverse cardiac events. In our high-risk population, patients with mild renal insufficiency undergoing PCI experience major adverse outcomes in the hospital and at long-term follow-up similar to those of patients with severe renal failure.

Treatment of the T98G glioblastoma cell line with antisense oligonucleotides directed toward mRNA encoding transforming growth factor-alpha and the epidermal growth factor receptor.

Antisense oligonucleotides (oligos) complementary to mRNA encoding transforming growth factor-alpha (TGF-alpha) and its target, the epidermal growth factor receptor (EGFR), are efficacious against human prostate and breast cancers carried in athymic nude mice. Glioblastomas, also regulated by EGFR expression, would appear to be similarly susceptible, and we now employ them against the T98G tumor model. T98G cells were distributed into wells and allowed to adhere prior to addition of oligos (12.5 microM) directed against TGF-alpha and/or EGFR for 6 d of treatment before thymidine radiolabeling. Supplemental media and oligos (25 microM final concentration) were added after d 3. Statistically significant inhibition by oligos directed against TGF-alpha, EGFR, and their combination was 13.8%, 26.3%, and 18.1%, respectively. In a subsequent experiment cells were incubated with increasing amounts of each oligo and their combination for 3 d prior to radiolabeling. Statistically significant inhibition of growth for either oligo at every concentration was found. Cells incubated with 6.25, 12.5, 25, and 50 microM antisense directed against TGF-alpha had a mean inhibition of 29.3%, 33.3%, 21.7%, and 46.6%, respectively. Cells similarly treated with oligos against EGFR had a mean inhibition of 77.9%, 80.3%, 82.0%, and 83.7%, respectively, and cells incubated with 6.25, 12.5, 25 and 50 microM of each oligo had a mean inhibition of 74.7%, 70.6%, 70.8%, and 76.3%, respectively. Lastly, in a paired experiment, cells treated with 0, 0.39, 0.78, 1.56, 3.125, and 6.25 microM of oligos, either specifically directed against EGFR or a random control, for 3 d were evaluated for both thymidine incorporation and EGFR expression. Statistically significant inhibition of 3H-thymidine incorporation was seen in cells with the oligo specifically directed against EGFR at 3.125 microM and 6.25 microM when compared to non-oligo containing controls. This was accompanied by a comparable significantly decreased expression of a low-MW reactive derivative of EGFR at 3.125 microM and 6.25 microM in Western blots, and of a high-MW reactive EGFR at 6.25 microM. The significant effect against high-MW EGFR was observed vs both the non-oligo containing control and the random sequence. Oligo concentrations between 0.78 and 1.5 microM also resulted in decreased expression of the low-MW form, but not significant differences in thymidine radiolabeling. In recovery experiments, cells treated initially with greater oligo concentrations required significantly increased time to recover, particularly in cells treated with EGFR directed oligos. Intracellular uptake and nuclear localization was demonstrated with FITC tagged oligos. In summary, even at relatively low oligo concentrations and short exposure, oligos against TGF-alpha, and particularly EGFR, significantly inhibit in vitro growth of the T98G glioblastoma, possibly mediated by decreased EGFR expression.

Biotinylation of antisense oligonucleotides does not alter lipofectin enhanced cellular uptake in prostate cancer cell lines.

Biotinylation is a common modification made to pharmaceuticals, including antisense oligonucleotides (oligos), to enhance their specific delivery. Such agents bind to targets that have been previously labeled with conjugated avidin, or alternatively, heteroconjugate monoclonal antibodies that have dual biotin and tumor-specific antigen specificities may be employed. However, for a drug to be efficacious it must also be taken up by the targeted cells. This is frequently difficult for large molecular weight compounds and cationic lipids, like lipofectin, are often employed. However, the effect of biotinylation on oligo uptake has not been examined in the presence of lipofectin, particularly in prostate cancer cells. Oligos conjugated with biotin and FITC were incubated in vitro with LNCaP and PC-3 cells in the presence of a previously determined effective concentration of lipofectin. Fluorescent uptake and distribution was compared to similar oligos that were not biotinylated. The results demonstrate that biotinylation does not alter the uptake of oligos in LNCaP or PC-3 prostate cancer cells, nor does it alter their retention or cytoplasmic distribution in PC-3 cells when used with lipofectin.

Changes in radical prostatectomy and radiation therapy rates for African Americans and whites.

There are racial differences in prostate cancer outcomes. One variable influencing end results is treatment for cure: either radical prostatectomy (RP) or radiation therapy (RT). The purpose of this report is to determine changes in diagnosis rates of localized prostate cancer between the years before prostate-specific antigen (PSA) use (1973-1988) and the years after PSA use (1989-1996), to evaluate differences in RP and RT rates between the pre-PSA and post-PSA eras, to assess differences in RP and RT rates between African Americans and whites between these intervals. The Surveillance, Epidemiology, and End Results (SEER) data were used and evaluated. Both African Americans and whites had statistically increased rates of localized prostate cancer diagnosed (70.4 and 49.0 in 1973 through 1988 and 123.1 and 84.9 in 1989 through 1996, respectively [p < 0.05]). The differences between the pre-PSA and post-PSA eras for African Americans and whites for RP (3.6 vs. 44.3 and 5.0 vs. 44.9, respectively) and RT (23.6 vs. 61.6 and 17.0 vs. 38.1, respectively) were all significant (p < 0.05). Both African Americans and whites had increased rates of RP from 3.6 and 5.0 to 44.3 and 44.9, respectively, and RT from 23.6 and 17.0 to 61.6 and 38.1 during the pre- and post-PSA years.