Hit proteins, mitochondria and cancer

The histidine triad (HIT) superfamily comprises proteins that share the histidine triad motif, His-ϕ-His-ϕ-His-ϕ-ϕ, where ϕ is a hydrophobic amino acid. HIT proteins are ubiquitous in prokaryotes and eukaryotes. HIT proteins bind nucleotides and exert dinucleotidyl hydrolase, nucleotidylyl transferase or phosphoramidate hydrolase enzymatic activity. In humans, 5 families of HIT proteins are recognized. The accumulated epidemiological and experimental evidence indicates that two branches of the superfamily, the HINT (Histidine Triad Nucleotide Binding) members and FHIT (Fragile Histidine Triad), have tumor suppressor properties but a conclusive physiological role can still not be assigned to these proteins. Aprataxin forms another discrete branch of the HIT superfamily, is implicated in DNA repair mechanisms and unlike the HINT and FHIT members, a defective protein can be conclusively linked to a disease, ataxia with oculomotor apraxia type 1. The scavenger mRNA decapping enzyme, DcpS, forms a fourth branch of the HIT superfamily. Finally, the GalT enzymes, which exert specific nucleoside monophosphate transferase activity, form a fifth branch that is not implicated in tumorigenesis. The molecular mechanisms by which the HINT and FHIT proteins participate in bioenergetics of cancer are just beginning to be unraveled. Their purported actions as tumor suppressors are highlighted in this review.

How reliable is Ki-67 immunohistochemistry in grade 2 breast carcinomas? A QA study of the Swiss Working Group of Breast- and Gynecopathologists

Adjuvant chemotherapy decisions in breast cancer are increasingly based on the pathologist's assessment of tumor proliferation. The Swiss Working Group of Gyneco- and Breast Pathologists has surveyed inter- and intraobserver consistency of Ki-67-based proliferative fraction in breast carcinomas. Methods Five pathologists evaluated MIB-1-labeling index (LI) in ten breast carcinomas (G1, G2, G3) by counting and eyeballing. In the same way, 15 pathologists all over Switzerland then assessed MIB-1-LI on three G2 carcinomas, in self-selected or pre-defined areas of the tumors, comparing centrally immunostained slides with slides immunostained in the different laboratoires. To study intra-observer variability, the same tumors were re-examined 4 months later. Results The Kappa values for the first series of ten carcinomas of various degrees of differentiation showed good to very good agreement for MIB-1-LI (Kappa 0.56–0.72). However, we found very high inter-observer variabilities (Kappa 0.04–0.14) in the read-outs of the G2 carcinomas. It was not possible to explain the inconsistencies exclusively by any of the following factors: (i) pathologists' divergent definitions of what counts as a positive nucleus (ii) the mode of assessment (counting vs. eyeballing), (iii) immunostaining technique, and (iv) the selection of the tumor area in which to count. Despite intensive confrontation of all participating pathologists with the problem, inter-observer agreement did not improve when the same slides were re-examined 4 months later (Kappa 0.01–0.04) and intra-observer agreement was likewise poor (Kappa 0.00–0.35). Conclusion Assessment of mid-range Ki-67-LI suffers from high inter- and intra-observer variability. Oncologists should be aware of this caveat when using Ki-67-LI as a basis for treatment decisions in moderately differentiated breast carcinomas.

Somatostatin receptor subtype 2A immunohistochemistry using a new monoclonal antibody selects tumors suitable for in vivo somatostatin receptor targeting

High overexpression of somatostatin receptors in neuroendocrine tumors allows imaging and radiotherapy with radiolabeled somatostatin analogues. To ascertain whether a tumor is suitable for in vivo somatostatin receptor targeting, its somatostatin receptor expression has to be determined. There are specific indications for use of immunohistochemistry for the somatostatin receptor subtype 2A, but this has up to now been limited by the lack of an adequate reliable antibody. The aim of this study was to correlate immunohistochemistry using the new monoclonal anti-somatostatin receptor subtype 2A antibody UMB-1 with the gold standard in vitro method quantifying somatostatin receptor levels in tumor tissues. A UMB-1 immunohistochemistry protocol was developed, and tumoral UMB-1 staining levels were compared with somatostatin receptor binding site levels quantified with in vitro I-[Tyr]-octreotide autoradiography in 89 tumors. This allowed defining an immunohistochemical staining threshold permitting to distinguish tumors with somatostatin receptor levels high enough for clinical applications from those with low receptor expression. The presence of >10% positive tumor cells correctly predicted high receptor levels in 95% of cases. In contrast, absence of UMB-1 staining truly reflected low or undetectable somatostatin receptor expression in 96% of tumors. If 1% to 10% of tumor cells were stained, a weak staining intensity was suggestive of low somatostatin receptor levels. This study allows for the first time a reliable recommendation for eligibility of an individual patient for in vivo somatostatin receptor targeting based on somatostatin receptor immunohistochemistry. Under optimal methodological conditions, UMB-1 immunohistochemistry may be equivalent to in vitro receptor autoradiography.

Evidence of prognostic relevant expression profiles of heat-shock proteins and glucose-regulated proteins in oesophageal adenocarcinomas

A high percentage of oesophageal adenocarcinomas show an aggressive clinical behaviour with a significant resistance to chemotherapy. Heat-shock proteins (HSPs) and glucose-regulated proteins (GRPs) are molecular chaperones that play an important role in tumour biology. Recently, novel therapeutic approaches targeting HSP90/GRP94 have been introduced for treating cancer. We performed a comprehensive investigation of HSP and GRP expression including HSP27, phosphorylated (p)-HSP27((Ser15)), p-HSP27((Ser78)), p-HSP27((Ser82)), HSP60, HSP70, HSP90, GRP78 and GRP94 in 92 primary resected oesophageal adenocarcinomas by using reverse phase protein arrays (RPPA), immunohistochemistry (IHC) and real-time quantitative RT-PCR (qPCR). Results were correlated with pathologic features and survival. HSP/GRP protein and mRNA expression was detected in all tumours at various levels. Unsupervised hierarchical clustering showed two distinct groups of tumours with specific protein expression patterns: The hallmark of the first group was a high expression of p-HSP27((Ser15, Ser78, Ser82)) and low expression of GRP78, GRP94 and HSP60. The second group showed the inverse pattern with low p-HSP27 and high GRP78, GRP94 and HSP60 expression. The clinical outcome for patients from the first group was significantly improved compared to patients from the second group, both in univariate analysis (p = 0.015) and multivariate analysis (p = 0.029). Interestingly, these two groups could not be distinguished by immunohistochemistry or qPCR analysis. In summary, two distinct and prognostic relevant HSP/GRP protein expression patterns in adenocarcinomas of the oesophagus were detected by RPPA. Our approach may be helpful for identifying candidates for specific HSP/GRP-targeted therapies.

Facile double-functionalization of designed ankyrin repeat proteins using click and thiol chemistries

Click chemistry is a powerful technology for the functionalization of therapeutic proteins with effector moieties, because of its potential for bio-orthogonal, regio-selective, and high-yielding conjugation under mild conditions. Designed Ankyrin Repeat Proteins (DARPins), a novel class of highly stable binding proteins, are particularly well suited for the introduction of clickable methionine surrogates such as azidohomoalanine (Aha) or homopropargylglycine (Hpg), since the DARPin scaffold can be made methionine-free by an M34L mutation in the N-cap which fully maintains the biophysical properties of the protein. A single N-terminal azidohomoalanine, replacing the initiator Met, is incorporated in high yield, and allows preparation of "clickable" DARPins at about 30 mg per liter E. coli culture, fully retaining stability, specificity, and affinity. For a second modification, we introduced a cysteine at the C-terminus. Such DARPins could be conveniently site-specifically linked to two moieties, polyethylene glycol (PEG) to the N-terminus and the fluorophore Alexa488 to the C-terminus. We present a DARPin selected against the epithelial cell adhesion molecule (EpCAM) with excellent properties for tumor targeting as an example. We used these doubly modified molecules to measure binding kinetics on tumor cells and found that PEGylation has no effect on dissociation rate, but slightly decreases the association rate and the maximal number of cell-bound DARPins, fully consistent with our previous model of PEG action obtained in vitro. Our data demonstrate the benefit of click chemistry for site-specific modification of binding proteins like DARPins to conveniently add several functional moieties simultaneously for various biomedical applications.

Intraoperative template-molded bone flap reconstruction for patient-specific cranioplasty

Cranioplasty is a common neurosurgical procedure. Free-hand molding of polymethyl methacrylate (PMMA) cement into complex three-dimensional shapes is often time-consuming and may result in disappointing cosmetic outcomes. Computer-assisted patient-specific implants address these disadvantages but are associated with long production times and high costs. In this study, we evaluated the clinical, radiological, and cosmetic outcomes of a time-saving and inexpensive intraoperative method to mold custom-made implants for immediate single-stage or delayed cranioplasty. Data were collected from patients in whom cranioplasty became necessary after removal of bone flaps affected by intracranial infection, tumor invasion, or trauma. A PMMA replica was cast between a negative form of the patient's own bone flap and the original bone flap with exactly the same shape, thickness, and dimensions. Clinical and radiological follow-up was performed 2 months post-surgery. Patient satisfaction (Odom criteria) and cosmesis (visual analogue scale for cosmesis) were evaluated 1 to 3 years after cranioplasty. Twenty-seven patients underwent intraoperative template-molded patient-specific cranioplasty with PMMA. The indications for cranioplasty included bone flap infection (56%, n = 15), calvarian tumor resection (37%, n = 10), and defect after trauma (7%, n = 2). The mean duration of the molding procedure was 19 ± 7 min. Excellent radiological implant alignment was achieved in 94% of the cases. All (n = 23) but one patient rated the cosmetic outcome (mean 1.4 years after cranioplasty) as excellent (70%, n = 16) or good (26%, n = 6). Intraoperative cast-molded reconstructive cranioplasty is a feasible, accurate, fast, and cost-efficient technique that results in excellent cosmetic outcomes, even with large and complex skull defects.

Orbital involvement in Pott's puffy tumor: a systematic review of published cases

Pott's puffy tumor (PPT) is a frontal subperiosteal abscess associated with underlying frontal bone osteomyelitis. It represents a well-known source of sinogenic intracranial infection, but the orbital complications related to this entity are rarely reported. The goal of this study was to characterize the orbital involvement in PPT.

Annexins as cell-type-specific markers in the developing chicken chorionallantoic membrane

Between day E8 and E12 of embryonic development, the chicken chorioallantoic membrane (CAM) undergoes massive structural rearrangement enabling calcium-uptake from the eggshell to supply the growing embryo. However, the contribution of the various cell types of the chorionic epithelium including the capillary covering (CC) cells, villus cavity (VC) cells, endothelial-like cells, and basal cells to this developmental program is largely unknown. In order to obtain markers for the different cell types in the chorionic epithelium, we determined the expression patterns of various calcium-binding annexins in the developing chicken CAM. By reverse transcription/polymerase chain reaction with primers deduced from nucleotide sequences available in various databases, the presence of annexin (anx)-1, anx-2, anx-5, and anx-6 was demonstrated at days E8 and E12. Quantitative immunoblotting with novel antibodies raised against the recombinant proteins revealed that anx-1 and anx-5 were significantly up-regulated at day E12, whereas anx-2 and anx-6 expression remained almost unchanged in comparison to levels at day E8. Immunohistochemistry of paraffin-embedded sections of E12 CAM revealed anx-1 in CC cells and VC cells. Anx-2 was localized in capillaries in the chorionic epithelium and in basal cells of the allantoic epithelium, whereas anx-6 was detected in basal cells or endothelial-like cells of the chorionic epithelium and in the media of larger vessels in the mesenchyme. A 2-day exposure of the CAM to a tumor cell spheroid resulted in strong proliferation of anx-1-expressing CC cells suggesting that these cells participate in the embryonic response to experimental intervention. Thus, annexins exhibit complementary expression patterns and represent appropriate cell markers for the further characterization of CAM development and the interpretation of results obtained when using CAM as an experimental model.

Ex vivo assessment of chemotherapy-induced apoptosis and associated molecular changes in patient tumor samples

BACKGROUND: There are inherent conceptual problems in investigating the pharmacodynamics of cancer drugs in vivo. One of the few possible approaches is serial biopsies in patients. However, this type of research is severely limited by methodological and ethical constraints. MATERIALS AND METHODS: A modified 3-dimensional tissue culture technique was used to culture human tumor samples, which had been collected during routine cancer operations. Twenty tumor samples of patients with non-small cell lung cancer (NSCLC) were cultured ex vivo for 120 h and treated with mitomycin C, taxotere and cisplatin. The cytotoxic activity of the anticancer agents was quantified by assessing the metabolic activity of treated tumor cultures and various assays of apoptosis and gene expression were performed. RESULTS: The proliferative activity of the tissue was maintained in culture as assessed by Ki-67 staining. Mitomycin C, cisplatin and taxotere reduced the metabolic activity of the tumor tissue cultures by 51%, 29% and 20%, respectively, at 120 h. The decrease in metabolic activity corresponded to the induction of apoptosis as demonstrated by the typical morphological changes, such as chromatin condensation and nuclear fragmentation. In addition, activated caspase-3 could be verified in apoptotic cells by immunohistochemistry. To verify functional aspects of apoptosis, the induction of chemotherapy-induced cell death was inhibited with the caspase inhibitor z-VAD.fmk. RNA was extracted from the tissue cultures after 120 h of ex vivo drug treatment and was of sufficient quality to allow quantitative PCR. CONCLUSION: The 3-dimensional ex vivo culture technique is a useful method to assess the molecular effects of pharmacological interventions in human cancer samples in vitro. This culture technique could become an important tool for drug development and for the prediction of in vivo drug efficacy.

Tumor necrosis factor-alpha: alternative role as an inhibitor of osteoclast formation in vitro

TNFalpha is known to stimulate the development and activity of osteoclasts and of bone resorption. The cytokine was found to mediate bone loss in conjunction with inflammatory diseases such as rheumatoid arthritis or chronic aseptic inflammation induced by wear particles from implants and was suggested to be a prerequisite for the loss of bone mass under estrogen deficiency. In the present study, the regulation of osteoclastogenesis by TNFalpha was investigated in co-cultures of osteoblasts and bone marrow or spleen cells and in cultures of bone marrow and spleen cells grown with CSF-1 and RANKL. Low concentrations of TNFalpha (1 ng/ml) caused a >90% decrease in the number of osteoclasts in co-cultures, but did not affect the development of osteoclasts from bone marrow cells. In cultures with p55TNFR(-/-) osteoblasts and wt BMC, the inhibitory effect was abrogated and TNFalpha induced an increase in the number of osteoclasts in a dose-dependent manner. Osteoblasts were found to release the inhibitory factor(s) into the culture supernatant after simultaneous treatment with 1,25(OH)(2)D(3) and TNFalpha, this activity, but not its release, being resistant to treatment with anti-TNFalpha antibodies. Dexamethasone blocked the secretion of the TNFalpha-dependent inhibitor by osteoblasts, while stimulating the development of osteoclasts. The data suggest that the effects of TNFalpha on the differentiation of osteoclast lineage cells and on bone metabolism may be more complex than hitherto assumed and that these effects may play a role in vivo during therapies for inflammatory diseases.

The selective Cox-2 inhibitor Celecoxib suppresses angiogenesis and growth of secondary bone tumors: an intravital microscopy study in mice

BACKGROUND: The inhibition of angiogenesis is a promising strategy for the treatment of malignant primary and secondary tumors in addition to established therapies such as surgery, chemotherapy, and radiation. There is strong experimental evidence in primary tumors that Cyclooxygenase-2 (Cox-2) inhibition is a potent mechanism to reduce angiogenesis. For bone metastases which occur in up to 85% of the most frequent malignant primary tumors, the effects of Cox-2 inhibition on angiogenesis and tumor growth remain still unclear. Therefore, the aim of this study was to investigate the effects of Celecoxib, a selective Cox-2 inhibitor, on angiogenesis, microcirculation and growth of secondary bone tumors. METHODS: In 10 male severe combined immunodeficient (SCID) mice, pieces of A549 lung carcinomas were implanted into a newly developed cranial window preparation where the calvaria serves as the site for orthotopic implantation of the tumors. From day 8 after tumor implantation, five animals (Celecoxib) were treated daily with Celecoxib (30 mg/kg body weight, s.c.), and five animals (Control) with the equivalent amount of the CMC-based vehicle. Angiogenesis, microcirculation, and growth of A549 tumors were analyzed by means of intravital microscopy. Apoptosis was quantified using the TUNEL assay. RESULTS: Treatment with Celecoxib reduced both microvessel density and tumor growth. TUNEL reaction showed an increase in apoptotic cell death of tumor cells after treatment with Celecoxib as compared to Controls. CONCLUSION: Celecoxib is a potent inhibitor of tumor growth of secondary bone tumors in vivo which can be explained by its anti-angiogenic and pro-apoptotic effects. The results indicate that a combination of established therapy regimes with Cox-2 inhibition represents a possible application for the treatment of bone metastases.

[Pulmonary microscopic tumor embolism syndrome]

HISTORY: A 76-year-old woman and a 62-year-old man were both referred to our clinic because of an unexplained weight loss, increasing dry cough and shortness of breath. INVESTIGATIONS: Investigations revealed an adenocarcinoma of the colon with retroperitoneal, mediastinal and supraclavicular lymph node metastasis and poorly differentiated carcinoma of the prostate with extensive bone metastases. During their hospital stay both patients developed increasing shortness of breath and clinical signs of right heart failure. Echocardiography confirmed severe pulmonary hypertension and dilatation of the right ventricle in both patients. Despite the high degree of clinical suspicion CT scans of the thorax could not demonstrate pulmonary embolism. DIAGNOSIS, TREATMENT AND COURSE: During the following days the patients condition deteriorated further and both patients' died from irreversible right heart failure. Both autopsies showed extensive metastatic adenocarcinoma with marked angiosis carcinomatosa of the lungs with numerous occlusions of small arteries and arterioles and resulting cor pulmonale. Thrombotic pulmonary embolism could not be detected. CONCLUSION: In patients with malignant neoplasms, especially adenocarcinomas, dyspnea and signs of increasing pulmonary artery pressure, the possibility of a microscopic pulmonary tumor embolism should be considered after exclusion of more usual causes especially thrombotic pulmonary embolism. In selected cases a cytologic examination of blood aspirated from a wedged pulmonary artery catheter can be performed to prove angiosis is carcinomatosa.

[Follow-up in a boy with Leydig cell tumor after selective surgery]

A 8(6/12) year-old-boy presented with precocious puberty and a slightly enlarged left testis. After a detailed examination a Leydig cell tumour was diagnosed. Surgical exploration revealed an encapsulated tumour, 2.7 cm in length, which was selectively removed without orchidectomy. Within one year the clinical signs of pubertal precocity disappeared, the bone age did not further advance and height velocity declined from 8.2 cm / year (+3.9 SDS) to 4.1 cm/year (-1.0 SDS). Physiologically, he entered puberty at the chronological age of twelve years, presenting at that age, in comparison to his peer group, a slightly decreased pubertal growth spurt. However, bearing in mind that being precocious in puberty he started in fact his pubertal growth spurt at a far earlier age, therefore, this acceleration of height before operation has to be added to the centimetres gained during pubertal development thereafter resuiting consequently in an absolute normal pubertal growth spurt. This underlines the fact that the individual growth spurt and, therefore, the total amount of centimetres gained is very much robust. Ten years later, the patient ended up well within his familial target height and remained free of disease. We report on a long-term follow-up of a prepubertal boy after testis-sparing surgery for Leydig-cell-tumour.

Retinal pigment epithelium damage enhances expression of chemoattractants and migration of bone marrow-derived stem cells

PURPOSE: To characterize chemoattractants expressed by the retinal pigment epithelium (RPE) after sodium iodate (NaIO3)-induced damage and to investigate whether ocular-committed stem cells preexist in the bone marrow (BM) and migrate in response to the chemoattractive signals expressed by the damaged RPE. METHODS: C57/BL6 mice were treated with a single intravenous injection of NaIO3 (50 mg/kg) to create RPE damage. At different time points real-time RT-PCR, ELISA, and immunohistochemistry were used to identify chemoattractants secreted in the subretinal space. Conditioned medium from NaIO3-treated mouse RPE was used in an in vitro assay to assess chemotaxis of stem cell antigen-1 positive (Sca-1+) BM mononuclear cells (MNCs). The expression of early ocular markers (MITF, Pax-6, Six-3, Otx) in migrated cells and in MNCs isolated from granulocyte colony-stimulating factor (G-CSF) and Flt3 ligand (FL)-mobilized and nonmobilized peripheral blood (PB) was analyzed by real-time RT-PCR. RESULTS: mRNA for stromal cell-derived factor-1 (SDF-1), C3, hepatocyte growth factor (HGF), and leukemia inhibitory factor (LIF) was significantly increased, and higher SDF-1 and C3 protein secretion from the RPE was found after NaIO3 treatment. A higher number of BMMNCs expressing early ocular markers migrated to conditioned medium from damaged retina. There was also increased expression of early ocular markers in PBMNCs after mobilization. CONCLUSIONS: Damaged RPE secretes cytokines that have been shown to serve as chemoattractants for BM-derived stem cells (BMSCs). Retina-committed stem cells appear to reside in the BM and can be mobilized into the PB by G-CSF and FL. These stem cells may have the potential to serve as an endogenous source for tissue regeneration after RPE damage.

Pattern of mineralization after regenerative periodontal therapy with enamel matrix proteins

A derivative (EMD) of enamel matrix proteins (EMPs) is used for periodontal regeneration because EMPs are believed to induce the formation of acellular extrinsic fiber cementum (AEFC). Other reports, however, indicate that EMPs have osteogenic potential. The aim of this study was to characterize the nature of the tissue that forms on the root surface following application of EMD. Ten human teeth affected by periodontitis and scheduled for extraction were treated with EMD. Four to six weeks later, they were extracted and processed for analysis by light microscopy and transmission electron microscopy. Immunocytochemistry with antibodies against bone sialoprotein (BSP) and osteopontin (OPN) was performed to determine the mineralization pattern. The newly formed tissues on the root were thick and contained embedded cells. Small mineralization foci were regularly seen, and large organic matrix patches were occasionally seen, but a distinct mineralization front was lacking. While labeling for BSP was always associated with small mineralization foci and large matrix patches, OPN labeling was seen inconsistently. It is concluded that tissues resembling either cellular intrinsic fiber cementum or a type of bone were observed. The mineralization pattern mostly resembled that found in bone, except for a few areas that exhibited a hitherto undescribed mineralization pattern.