Difference in Risk Factors for Breast Cancer by ER Status in an Indigenous African Population.

Introduction. Breast cancer is the commonest cancer among women globally. In Uganda, it is on the rise, projected at a 4.5% annual ASR increase (age standardized incidence rate). The reasons for this steep increase are not fully established. In the recent past, gene profiling in tumor tissues suggests that breast cancers are divided into subtypes dependent on the presence or absence of oestrogen receptor, progesterone, and human epidermal growth factor receptor 2 (HER 2). These subtypes do have distinctive clinical outcomes and perhaps risk factors from past studies. There is paucity of data on hormonal receptor status and the traditionally known risk factors in sub-Saharan Africa. The purpose of this study therefore was to establish the differences between ER status and the traditionally known risk factors for breast cancer in Uganda. Methods. An observational analytical hospital, based study, carried out at Makerere University, College of Health Sciences. Formalin fixed and paraffin imbedded sections were prepared for haemotoxylin and eosin (H&E) stains and immunohistochemistry (IHC). Ethical approval was obtained. Results. A total of 113 women were recruited. Mean age was 45 years (SD14). There were no significant differences in selected risk factors (setting, age, contraceptive use, parity, breast feeding, or menarche) by ER status although ER negative tumors had significantly higher grade tumors (by a factor of two) compared to ER positive tumors. Conclusion. There were no significant differences among risk factors by ER status contrary to what several other studies suggest. The manifestation of breast cancer in Africa warrants further extensive inquiry.

Role of human and mouse HspB1 in metastasis.

Heat shock proteins (HSP) are a group of physiologically-essential, highly-conserved proteins that are induced by heat shock, as well as by other environmental and pathophysiological stressors. The twentyseven kDa heat shock protein (Hsp27; HspB1) is highly expressed in tumor tissues of patients diagnosed with cancer and expression levels correlate with poor prognosis. HspB1 plays a dual role in cancer and promotes both cancer development by suppressing host anti-cancer response, such as apoptosis and senescence, and facilitates the enhanced expression of metastastic genes. HspB1-mediated protection from tumor cell apoptosis induced by chemotherapeutic drugs occurs through several mechanisms, including decreased production of reactive oxygen species, restoration of protein homeostasis and promotion of cell survival by protein folding, stabilization of actin-cytoskeleton, delayed release of cytochrome c from mitochondria and inhibition of activation of caspase-3. High levels of HSP expression affect tumor susceptibility to adjuvant cancer treatments, including chemotherapy, hyperthermia, and radiation. This review highlights the most recent findings and role of HspB1 in metastasis.

Development of an XTT tetrazolium salt-based assay for detection of specific hyperthermia sensitizers in a high-flux screening programme.

It is now possible to search for new drugs using high-throughput screening of chemical libraries accumulated over the past few years. To detect potential new hyperthermia sensitizers, we are screening for chemical inhibitors of thermotolerance. For the screening of a large chemical library, a rapid and simple assay based on the XTT-tetrazolium salt with the addition of intermediate electron acceptor, phenazine methosulphate (PMS) as a promoter, was developed. It was found that the sensitivity of the XTT/PMS assay is sufficient for assessing thermal cell killing and thermotolerance, although it was highly dependent on cell number and type. When the formazan assay system was challenged with the bioflavonoid drug quercetin (up to 25mm) and validated against the clonogenic cell survival assay, significant decreases in thermotolerant cell viability were observed, directly reflecting inhibition of thermotolerance. Although short-term assays can, in some instances, underestimate overall cell killing, the dose dependency of inhibition of thermotolerance by quercetin recorded in this study by clonogenic and XTT/PMS assays was similar. Application of the XTT/PMS assay in chemical library screening was highly effective in differentiating potential thermotolerance inhibitors from both chemicals with lack of efficacy and from toxic compounds. Taken together, these results show that the XTT/PMS assay, when carried out under careful conditions, is well suited for primary high-flux screen of many thousands of compounds, thus opening up new areas for discovery of hyperthermia sensitizers.

Targeting HSP70-induced thermotolerance for design of thermal sensitizers.

Thermal therapy has been shown to be an extremely powerful anti-cancer agent and a potent radiation sensitizer. However, the full potential of thermal therapy is hindered by a number of considerations including highly conserved heat resistance pathways in tumour cells and inhomogeneous heating of deep-seated tumours due to energy deposition and perfusion issues. This report reviews recent progress in the development of hyperthermia sensitizing drugs designed to specifically amplify the effects of hyperthermia. Such agents might be particularly useful in situations where heating is not adequate for the full biological effect or is not homogeneously delivered to tumours. The particular pathway concentrated on is thermotolerance, a complex, inducible cellular response that leads to heat resistance. This paper will concentrate on the molecular pathways of thermotolerance induction for designing inhibitors of heat resistance/thermal sensitizers, which may allow the full potential of thermal therapy to be utilized.

Cyclooxygenase inhibitors are potent sensitizers of prostate tumours to hyperthermia and radiation.

It has previously been demonstrated that hyperthermia can activate prostaglandin synthesis and that prostaglandins are protective against hyperthermia. This study examined the use of inhibitors of prostaglandin synthesis on the response of prostate tumours to hyperthermia. The non-steroidal anti-inflammatory drugs (NSAID) ibuprofen and sulindac, known cyclooxygenase inhibitors that inhibit prostaglandin production, were effective hyperthermia sensitizers and augmented growth delay of DU-145 and PC-3 prostate tumours to combined radiation and hyperthermia treatment protocols. Pre-treatment of mice with ibuprofen and sulindac at hyperthermia sensitizing doses resulted in significant (p < 0.01) inhibition of hyperthemia-induced serum prostaglandin E2. These findings indicate that NSAID may have both sensitizing effects on prostate tumour growth and may function by inhibiting prostaglandin synthesis.

Effects of the flavonoid drug quercetin on the response of human prostate tumours to hyperthermia in vitro and in vivo.

Tumour hyperthermia, although potentially a powerful therapeutic agent and radiation sensitizer, is hindered by a number of considerations including inhomogeneous heating of deep seated tumours due to energy deposition and perfusion issues. One solution is to design hyperthermia sensitizers to amplify the effects of hyperthermia, particularly at cold spots within the tumour undergoing treatment. This study examined the use of Quercetin, a flavonoid drug shown previously to antagonize the expression of HSP72 and induce apoptosis as a sensitizer of prostate cancer growth in vivo. Quercetin dose-dependently suppressed PC-3 tumour growth in vitro and in vivo. When combined in a treatment protocol with hyperthermia, quercetin drastically inhibited tumour growth and potently amplified the effects of hyperthermia on two prostate tumour types, PC-3 and DU-145 in vivo. These experiments, thus, suggest the use of Quercetin as a hyperthermia sensitizer in the treatment of prostate carcinoma.

Role of calcium activated kinases and phosphatases in heat shock factor-1 activation.

HSF-1 is regulated at multiple molecular levels through intra- and intermolecular protein-protein interactions as well as by post-translational modification through phosphorylation. We have found that elevating intracellular calcium ion levels by exposure to the ionophore A23187 or thapsigargin inhibits the conversion of HSF-1 from a latent cytoplasmic form to its nuclear/DNA binding form. To examine a role for calcium/calmodulin regulated enzymes in this process, we examined the ability of specific inhibitors to abrogate the effects of calcium elevation. While the inhibitor of calmodulin dependent kinase II, KCN62 enhanced activation of HSF-1 during heat shock, it failed to block the inhibitory effects of calcium increase. By contrast, the immunosuppresant drugs cyclosporin A and FK506 abolished the effects of calcium elevation on HSF-1 activation. As the biological effects of the drugs are effected through inhibition of the calcium/calmodulin regulated phosphatase calcineurin, this suggests a role for calcineurin in antagonizing HSF-1 activity. The experiments suggest the existence of phosphorylated residue(s) in HSF-1 important in one or more of the processes that lead to activation (trimerization, nuclear localization, DNA binding) and which becomes dephosphorylated due to the activation of a calcium/calmodulin/calcineurin complex.

HSP70 stimulates cytokine production through a CD14-dependant pathway, demonstrating its dual role as a chaperone and cytokine.

Here, we demonstrate a previously unknown function for the 70-kDa heat-shock protein (HSP70) as a cytokine. HSP70 bound with high affinity to the plasma membrane, elicited a rapid intracellular calcium flux, activated nuclear factor (NF)-kappaB and upregulated the expression of pro-inflammatory cytokines tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta and IL-6 in human monocytes. Furthermore, two different signal transduction pathways were activated by exogenous HSP70: one dependent on CD14 and intracellular calcium, which resulted in increased IL-1beta, IL-6 and TNF-alpha; and the other independent of CD14 but dependent on intracellular calcium, which resulted in an increase in TNF-alpha but not IL-1beta or IL-6. These findings indicate that CD14 is a co-receptor for HSP70-mediated signaling in human monocytes and are indicative of an previously unrecognized function for HSP70 as an extracellular protein with regulatory effects on human monocytes, having a dual role as chaperone and cytokine.

Mutation detection in the human HSP7OB' gene by denaturing high-performance liquid chromatography.

Variances, particularly single nucleotide polymorphisms (SNP), in the genomic sequence of individuals are the primary key to understanding gene function as it relates to differences in the susceptibility to disease, environmental influences, and therapy. In this report, the HSP70B' gene is the target sequence for mutation detection in biopsy samples from human prostate cancer patients undergoing combined hyperthermia and radiation therapy at the Dana-Farber Cancer Institute, using temperature-modulated heteroduplex analysis (TMHA). The underlying principles of TMHA for mutation detection using DHPLC technology are discussed. The procedures involved in amplicon design for mutation analysis by DHPLC are detailed. The melting behavior of the complete coding sequence of the target gene is characterized using WAVEMAKER software. Four overlapping amplicons, which span the complete coding region of the HSP70B' gene, amenable to mutation detection by DHPLC were identified based on the software-predicted melting profile of the target sequence. TMHA was performed on PCR products of individual amplicons of the HSP70B' gene on the WAVE Nucleic Acid Fragment Analysis System. The criteria for mutation calling by comparing wild-type and mutant chromatographic patterns are discussed.

HSP70 peptidembearing and peptide-negative preparations act as chaperokines.

We recently elucidated a novel function for the 70-kDa heat shock protein (HSP70) as a chaperone and a cytokine, a chaperokine in human monocytes. Here we show that peptide-bearing and peptide-negative HSP70 preparations isolated from EMT6 mammary adenocarcinoma cells (EMT6-HSP70) act as chaperokines when admixed with murine splenocytes. EMT6-HSP70 bound with high affinity to the surface of splenocytes recovered from naive BALB/c mice. The [Ca2+]i inhibitor BAPTA dose dependently inhibited HSP70- but not LPS-induced NF-kappaB activity and subsequent augmentation of proinflammatory cytokine TNF-alpha, IL-1beta, and IL-6 production. Taken together, these results suggest that presence of peptide in the HSP70 preparation is not required for spontaneous activation of cells of the innate immune system.

HSP70 and heat shock factor 1 cooperate to repress Ras-induced transcriptional activation of the c-fos gene.

Heat shock protein 70 (HSP70) is a molecular chaperone involved in protein folding and resistance to the deleterious effects of stress. Here we show that HSP70 suppresses transcription of c-fos, an early response gene that is a key component of the ubiquitous AP-1 transcription factor complex. HSP70 repressed Ras-induced c-fos transcription only in the presence of functional heat shock factor1 (HSF1). This suggests that HSP70 functions as a corepressor with HSF1 to inhibit c-fos gene transcription. Therefore, besides its known function in the stress response, HSP70 also has the property of a corepressor and combines with HSF1 to antagonize Fos expression and may thus impact multiple aspects of cell regulation.

Tumor suppressive action of indomethacin is NK-cell-independent.

This study was undertaken to determine whether NK-cells constitute a necessary mediator for the suppression of tumor growth by indomethacin. C57Bl mice with a methylcholantrene (MCG 101) tumor were studied. Indomethacin treatment was provided by daily subcutaneous injections (1 microgram/g body weight). NK-cells were depleted by treatment with a monoclonal antibody to NK1.1. Consecutive indomethacin injections prolonged survival in tumor bearing animals. Indomethacin was equally effective in animals with intact NK-cells as in NK-cell-depleted animals. Further, the MCG cells were apparently insensitive to the lytic activity of NK-cells in vivo. Thus, the clearance of intravenously injected MCG cells from lungs was not affected by depletion of NK-cells in vivo; in contrast, the corresponding clearance of NK-cell-sensitive YAC-1 lymphoma cells was strikingly reduced by the depletion of NK-cells. Our data suggest that NK cells are not a necessary mediator for the suppression of tumor growth by indomethacin.

Duration and mechanisms of the increased natural cytotoxicity seen after chronic voluntary exercise in rats.

We have recently shown that in vivo natural cytotoxicity is enhanced after chronic exercise in spontaneously hypertensive rats (SHRs). In the present report, we have studied the duration of this augmentation and some possible mechanisms involved. Exercise consisted of voluntary running for 4-5 weeks, with the running distance ranging from 2.7-15.6 km day(-1) during the last week of running. In vivo cytotoxicity was measured as clearance of injected 51Cr-labelled YAC-1 lymphoma cells from the lungs. The in vivo natural cytotoxicity was increased in running SHRs, and also in SHRs that had their running wheel locked for 24 and 48 h prior to the experiment, and was still present after 96 h. The enhancement of in vivo cytotoxicity after 5 weeks of exercise was abolished after an acute injection of the beta-adrenergic receptor antagonist timolol (0.5 mg kg(-1) i.v.), indicating that catecholamines are involved in this augmentation. Interestingly, 24 h after the last exercise bout, the increased natural cytotoxicity could be blocked by timolol. The opioid receptor antagonist naloxone given subcutaneously for 7 days by osmotic pumps (6 mg kg(-1) h(-1)) could not reverse the increased in vivo cytotoxicity seen in the running SHRs, suggesting that opioid receptor mechanisms are not involved, or at least not the naloxone-sensitive mu-receptor. Natural immunity was not influenced by the histamine H2 receptor antagonist ranitidine, either in controls or in runners, indicating that the natural killer cell-regulatory effect of histamine is not present in SHRs and does not seem to be involved in the exercise-induced changes in natural immune function. We conclude that the augmentation of in vivo natural cytotoxicity after voluntary chronic exercise in rats is long-lasting and that the augmentation is partly mediated by beta-adrenergic receptors.

Histaminergic regulation of interferon-gamma (IFN-gamma) production by human natural killer (NK) cells.

Monocytes, recovered from human peripheral blood by counter-current centrifugal elutriation, effectively inhibit the production of IFN-gamma by CD3-/56+ NK cells in response to IL-2. This study aimed at defining the nature of the inhibitory signal, particularly the importance of monocyte-derived reactive metabolites of oxygen. It was found that monocytes recovered from patients with chronic granulomatous disease (CGD), a condition characterized by deficient NADPH-oxidase activity of phagocytes, did not inhibit IFN-gamma production by NK cells. Further, catalase, a scavenger of hydrogen peroxide, completely reversed the inhibitory signal whereas scavengers of the superoxide anion, hypohalous acids, the hydroxyl radical, or nitric oxide synthesis inhibitors such as L-NMMA were ineffective. Inhibition of IFN-gamma production was operating on a pretranslational level, as indicated by the inability of enriched NK cells to accumulate IFN-gamma mRNA in the presence of elutriated monocytes. Hydrogen peroxide, at micromolar concentrations, reconstituted the inhibition of IFN-gamma production when added to enriched NK cells. Histamine, a biogenic amine which inhibits the generation of reactive oxygen metabolites in monocytes, abrogated the inhibition of IFN-gamma production in NK cells; by this mechanism, histamine strongly synergized with IL-2 to induce IFN-gamma in mixtures of NK cells and monocytes. The synergizing effect of histamine was specifically mediated by H2-type histamine receptors. We conclude that: (i) the induction of IFN-gamma mRNA in NK cells is effectively down-regulated by products of the oxidative metabolism of monocytes; and (ii) histamine effectively enhances IFN-gamma production by preventing monocyte-induced oxidative damage to NK cells.

Acute mental stress but not enforced muscle activity transiently increases natural cytotoxicity in spontaneously hypertensive rats.

The influence of acute mental stress and the effect of electrically induced skeletal muscle contractions on natural cytotoxicity in vivo was investigated in spontaneously hypertensive rats Natural cytotoxicity in vivo was measured as the clearance of injected 51Cr-labelled YAC-1 lymphoma cells from the lungs, which are specifically lysed by natural killer cells. The mental stress consisted of an air jet directed towards the animals in their cage for 25 min. During the mental stress there was a significant increase in natural cytotoxicity. Thus, retained radioactivity in the lungs was decreased to 74 +/- 6% of the control levels which was set to 100% (P < 0.01). This augmentation of YAC-1-cell clearance could be blocked with the beta-adrenergic receptor antagonist Timolol. Two hours after termination of the air stress, in vivo cytotoxicity had returned to control levels. In contrast, acute physical stress, consisting of electrically induced muscle contractions for 60 min, had no significant effects on in vivo cytotoxicity, either during the stimulation or 1, 2 or 24 h after the stimulation. Further, significantly increased plasma levels of adrenaline were seen after the air jet stress, but not after muscle stimulation. There were no significant changes in plasma noradrenaline levels either after air stress or muscle stimulation. These results indicate that changes in in vivo cytotoxicity after mild mental stress are dependent on increased plasma catecholamine levels while acute physical stress without changes in catecholamine levels, does not influence in vivo cytotoxicity.

Voluntary chronic exercise augments in vivo natural immunity in rats.

The effect of chronic voluntary exercise on the immune response was studied in spontaneously hypertensive rats. Exercise consisted of voluntary running in wheels for 5 wk, and the mean running distance was 4.2 km/24 h. In vivo cytotoxicity was measured as clearance of injected 51Cr-labeled YAC-1 lymphoma cells from the lungs. The clearance of YAC-1 cells in vivo was significantly increased in runners compared with sedentary controls (P < 0.001). The total number of mononuclear cells in the spleen was significantly decreased in runners compared with controls. Analysis of splenic lymphocyte phenotypes revealed a significantly increased fraction of OX52+/CD5- natural killer cells in runners compared with sedentary controls. In contrast to changes in natural immunity, immunoglobulins G and M levels in serum, the antibody response to antigen in vivo, and the proliferation of splenic T cells in vitro were unchanged. Our data suggest that chronic voluntary exercise augments natural cytotoxicity mechanisms in vivo, whereas splenic T-cell proliferation and the antibody-mediated immune response remain unchanged.

Chronic intracerebroventricular administration of beta-endorphin augments natural killer cell cytotoxicity in rats.

We have studied the effect of chronic intracerebroventricular (i.c.v.) infusion of different opioid peptides on natural killer (NK) cell mediated cytotoxicity in vivo in the spontaneously hypertensive rat (SHR). The in vivo NK cell activity was measured as the clearance of 51Cr-labelled YAC-l lymphoma cells from the lung tissues. Further, the phenotype of lymphocytes in spleen and peripheral blood was analysed by flow cytometry (FACS). All opioid drugs were administered i.c.v. for 6 days with osmotic minipumps releasing 1.0 microliter/h. beta-Endorphin (10 or 20 micrograms/rat per day) significantly increased NK cell cytotoxicity in vivo. The opioid receptor antagonist naloxone (10 mg/kg, i.p.) given immediately before the injection of YAC-lymphoma cells, completely abolished the effects of i.c.v. administered beta-endorphin. Corresponding doses of beta-endorphin administered subcutaneously (s.c.) with minipumps for 6 days did not significantly affect NK cell cytotoxicity. Neither Leu- or Met-enkephalin (20 micrograms/rat per day) nor dynorphin (20 micrograms/rat per day) administered i.c.v. had any significant effects on NK cell activity. In beta-endorphin treated SHR, the percentage of cells with NK cell phenotype (OX52+/CD5-) in peripheral blood was not significantly different from that of controls, while the percentage of cells with T cell phenotype (CD5+/OX52-) was significantly decreased. The percentage of splenic NK cells (OX52+/CD5-) and T cells (CD5+/OX52-) was also unchanged by beta-endorphin treatment i.c.v. These results suggest that of the opioid peptides administered i.c.v., only beta-endorphin augments in vivo NK cell mediated cytotoxicity. We thus conclude that these effects most probably are centrally and opioid receptor mediated effects, since beta-endorphin in the same dose administered peripherally does not influence in vivo NK cell cytotoxicity.

Natural immunity and chronic exercise in rats. The involvement of the spleen and the splenic nerves.

We have previously shown that voluntary running for 4-5 weeks in the spontaneously hypertensive rat (SHR) significantly increased in natural cytotoxic mechanism in vivo, measured as clearance of 51Cr YAC-1 lymphoma cells from the lungs. In the present study, we have studied the possible role of the spleen and the splenic nerves in this augmentation. The SHR were randomly allocated to either a voluntary exercise group or a sedentary control group. After four weeks of exercise the runners and sedentary control SHR were further assigned to one of four groups: 1) no surgery, 2) sham operation, 3) splenic nerve section and 4) splenectomy. Splenectomy drastically reduced in vivo cytotoxicity in both runners and sedentary controls, but in vivo cytotoxicity of splenectomized voluntary runners was significantly higher than that of splenectomized sedentary control animals. Selective denervation of the spleen did not affect the in vivo cytotoxicity. These results indicate that the enhanced in vivo natural cytotoxic mechanism following voluntary chronic exercise in SHR is partly dependent on intact splenic function. However, this enhancement does not seem to be mediated by the splenic sympathetic nerves.

Rapid detection of thymidylate synthase gene expression levels by semi-quantitative competitive reverse transcriptase polymerase chain reaction followed by quantitative digital image analysis.

We describe a simplified and reliable polymerase chain reaction (PCR) method to quantify thymidylate synthase (TS) gene expression levels from clinical human tumor biopsy samples as small as 100 mg using the beta-actin housekeeping gene as a reference standard. The semiquantitative RT-PCR is carried out by the coamplification of the target template and an external competitor using primer pairs common to both templates in the same reaction vessel. Quantitative digital image analysis is performed directly after electrophoresis, thus mRNA quantification is done quickly and without the use of radioactive substances. The observed relative TS gene expression levels varied between 3- and 40-fold, but most of the values were grouped within a 10-fold range. There is an observed 89% correlation between TS mRNA expression and protein levels. These findings suggest that preliminary experiments used to determine the linear range of RT-PCR amplification in non-competitive semiquantitative PCR experiments, and the use of radioactive substances to quantify PCR products may be unnecessary.

Histaminergic regulation of natural killer cell-mediated clearance of tumour cells in mice.

Treatment of Swiss albino mice with histamine enhanced the clearance of natural killer (NK)-cell sensitive YAC-1 lymphoma and B16/F10 melanoma cells from lung tissue in vivo, but did not affect the elimination of NK-cell-insensitive P815 mastocytoma cells. The effect of histamine was apparently mediated by H2-type histamine receptors (H2R) since it was blocked by ranitidine, and H2R antagonist. Histamine did not affect clearance of tumour cells in animals depleted of NK cells in vivo by treatment with antibodies to asialo-GM1 or NK1.1. The effect of histamine was time-dependent: pretreatment with histamine for 3 h significantly augmented the clearance of YAC-1 cells, whereas, pretreatment with histamine for 5 min was ineffective. Histamine potentiated the anti-tumour properties of NK-cell activators such as interleukin-2 (IL-2) or interferon-alpha (IFN-alpha) in vivo. None of these lymphokines significantly affected the clearance of YAC-1 cells unless animals were concomitantly treated with histamine. Treatment with ranitidine alone reduced the in vivo clearance of YAC-1 cells from lungs but did not affect the clearance of NK-cell-insensitive P815 cells. Effects of ranitidine on NK-cell function in vivo were not shared by a chemical control to ranitidine, AH20239AA, thus indicating that the inhibition of NK-cells results from H2R antagonism rather than non-specific toxicity. It is concluded that histaminergic mechanisms may be involved in the regulation of NK cell function in vivo.