Human carcinoma cell lines xenografted in athymic mice: biological and antigenic characteristics of an intraabdominal model

Summary In order to investigate in vivo clinical applications of murine monoclonal antibodies directed against human ovarian carcinoma a preclinical in vivo model was developed using BALB/c athymic mice. Three human carcinoma cell lines (MCF7, HT29, and SW626) were injected into the peritoneal cavity of pristane-primed animals and the biological and antigenic characteristics of the i.p. grown tumors were studied. The animals were killed when moribund or 6–8 weeks after tumor injection. At autopsy tumor take was observed in 85% of the injected animals, whereas palpable nodules were evident in only 83%. Examination of the peritoneal cavity revealed intraabdominal carcinomatosis with tumor masses varying in size between 0.2 and 0.5 cm in diameter and tumor sheets. The most frequently affected organs were the diaphragm, the liver, and the reproductive system. Ascitic fluid formation was rare and no animal developed tumors outside the peritoneal cavity. To determine whether the in vivo tumors retained the same antigenic characteristics as the in vitro cell lines, four monoclonal antibodies (MBrl, MOv2, MOv8, and MOv15) directed against ovarian carcinoma-associated antigens and two different experimental approaches (immunofluorescence and immunoblotting) were used. Variations at either a quantitative or a qualitative level were observed for some antigens, whereas no evident changes were apparent for others. In particular, the antigens detected by MBr1 and MOv15 on the MCF7 line both maintained high levels of expression and immunoblotting staining pattern, whereas the antigens detected by MOv2 on the HT29 and SW626 lines, although present at a high level, clearly changed their staining pattern. As regards the antigens recognized by MOv8 and MOv15 on the HT29 and SW626 lines, we observed a drastic decrease in the level of their expression and in many cases a drop below the threshold of detectability of the test. The intraabdominal carcinomatosis described partially mimics the growth characteristics of human ovarian cancer and maintains the expression of some antigenic markers associated with epithelial tumors of the ovary and may therefore be useful in devising immunodiagnostic and/or immunotherapeutic strategies for ovarian carcinoma.     

Structural analysis of turkey tendon collagen upon removal of the inorganic phase

Calcified leg flexor tendons in which the inorganic phase content had been lowered by progressive demineralization were studied by small angle X-ray diffraction and thermogravimetry. The X-ray diffraction results agree very well with the data previously obtained on calcified turkey tendon indicating that the method used to decalcify tendons provides good correspondence with the process of calcification. Up to five thermal processes can be detected in the thermogravimetric scans: (1) water release; (2) collagen decomposition; (3 and 4) combustion of the residual organic components; (5) carbonate removal from the apatitic phase. The temperature of collagen decomposition decreases at lower inorganic phase content in agreement with the higher thermal stability of calcified collagen fibrils compared with uncalcified ones. The decrease of collagen thermal stability upon decalification is paralleled by a decrease of the structural order of the collagen fibrils as indicated by small angle X-ray diffraction data. Decalcification down to about 40% wt of inorganic phase does not significantly alter the inorganic blocks that are regularly arranged inside the gap zone of the collagen. Further removal of inorganic phase down to about 15% wt provokes a variation of the intensity distribution of the small angle meridional reflections that can be ascribed to a reduction of the mean height of the inorganic blocks. At inorganic phase contents below 15% wt the gap region is more free to contract upon air drying as a result of the reduction of the mean length of the inorganic blocks.     

Effects of pH and ionic strength on the structure of collagen fibrils

The roles of pH and ionic strength on the structure and stability of collagen fibrils have been investigated by means of x-ray and neutron diffraction techniques. High-angle x-ray diffraction shows that a salt concentration of 0.5M KCl is sufficient to reduce the osmotic swelling and related disordering in the pH range 1–3. The relative intensities of the low-angle meridional x-ray and neutron diffraction Bragg reflections vary with pH. Difference Fourier syntheses between pH 7 and 1.6 data indicate, for both x-ray and neutron diffraction, a reduced scattering contribution from the telopeptides at low pH. Lyotropic relaxation is a crucial step in the appearance at low pH of a doubling of the 668-Å axial periodicity (D) of collagen fibrils. These results suggest that electrostatic interactions are essential for the structural stability of the telopeptide regions and of the 1D and 3D intermolecular staggers between collagen molecules.     

Calcium ions and osteoclastogenesis initiate the induction of bone formation by coral‐derived macroporous constructs

Coral-derived calcium carbonate/hydroxyapatite macroporous constructs of the genus Goniopora with limited hydrothermal conversion to hydroxyapatite (7% HA/CC) initiate the induction of bone formation. Which are the molecular signals that initiate pattern formation and the induction of bone formation? To evaluate the role of released calcium ions and osteoclastogenesis, 7% HA/CC was pre-loaded with either 500 μg of the calcium channel blocker, verapamil hydrochloride, or 240 μg of the osteoclast inhibitor, biphosphonate zoledronate, and implanted in the rectus abdominis muscle of six adult Chacma baboons Papio ursinus. Generated tissues on days 15, 60 and 90 were analysed by histomorphometry and qRT-PCR. On day 15, up-regulation of type IV collagen characterized all the implanted constructs correlating with vascular invasion. Zoledronate-treated specimens showed an important delay in tissue patterning and morphogenesis with limited bone formation. Osteoclastic inhibition yielded minimal, if any, bone formation by induction. 7% HA/CC pre-loaded with the Ca⁺⁺ channel blocker verapamil hydrochloride strongly inhibited the induction of bone formation. Down-regulation of bone morphogenetic protein-2 (BMP-2) together with up-regulation of Noggin genes correlated with limited bone formation in 7% HA/CC pre-loaded with either verapamil or zoledronate, indicating that the induction of bone formation by coral-derived macroporous constructs is via the BMPs pathway. The spontaneous induction of bone formation is initiated by a local peak of Ca⁺⁺ activating stem cell differentiation and the induction of bone formation.     

EGFR through STAT3 modulates ΔN63α expression to sustain tumor‐initiating cell proliferation in squamous cell carcinomas

Many squamous cell carcinomas (SCCs) are characterized by high levels of EGFR and by overexpression of the ΔNp63α isoform. Here, we investigated the regulation of ΔNp63α expression upon EGFR activation and the role of the EGFR–ΔNp63α axis in proliferation of SCC tumor‐initiating cells (TICs). SCC cell lines A‐431, Cal‐27, and SCC‐25 treated with EGF showed a time‐dependent increase in ΔNp63α expression at the protein and mRNA levels, which was blocked by the tyrosine kinase inhibitor (TKI) Lapatinib. RNA interference experiments suggested the role of STAT3 in regulating ΔNp63α expression downstream of EGFR. Inactivation of EGFR by the monoclonal antibody Cetuximab and RNA interference against STAT3 or ΔNp63α impaired the TICs ability to grow under non‐differentiating conditions. Radiation treatment, which triggers EGFR activation, induced ΔNp63α accumulation without affecting TICs proliferation, whereas the combination Cetuximab plus radiation significantly reduced TICs growth under non‐differentiating conditions. Together, our findings provide evidence that ΔNp63α expression is regulated by EGFR activation through STAT3 and that the EGFR–ΔNp63α axis is crucial for proliferation of TICs present in SCCs. J. Cell. Physiol. 228: 871–878, 2013. © 2012 Wiley Periodicals, Inc.     

Dual coenzyme specificity of photosynthetic glyceraldehyde-3-phosphate dehydrogenase interpreted by the crystal structure of A4 isoform complexed with NAD

Photosynthetic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of Spinacia oleracea belongs to a wide group of GAPDHs found in most organisms displaying oxygenic photosynthesis, including cyanobacteria, green and red algae, and higher plants. As a major catalytic difference with respect to glycolytic GAPDH, photosynthetic GAPDH exhibits dual cofactor specificity toward pyridine nucleotides with a preference for NADP(H). Here we report the crystal structure of NAD-complexed recombinant A(4)-GAPDH (NAD-A(4)-GAPDH) from Spinacia oleracea, expressed in Escherichia coli. Its superimposition onto native A(4)-GAPDH complexed with NADP (NADP-A(4)-GAPDH) pinpoints specific conformational changes resulting from cofactor replacement. In photosynthetic NAD-A(4)-GAPDH, the side chain of Asp32 is oriented toward the coenzyme to interact with the adenine ribose diol, similar to glycolytic GAPDHs (NAD-specific). On the contrary, in NADP-A(4)-GAPDH Asp32 moves away to accommodate the additional 2'-phosphate group of the coenzyme and to minimize electrostatic repulsion. Asp32 rotation is allowed by the presence of the small residue Ala40, conserved in most photosynthetic GAPDHs, replacing bulky amino acid side chains in glycolytic GAPDHs. While in NADP-A(4)-GAPDH two amino acids, Thr33 and Ser188, are involved in hydrogen bonds with the 2'-phosphate group of NADP, in the NAD-complexed enzyme these interactions are lacking. The crystallographic structure of NAD-A(4)-GAPDH highlights that four residues, Thr33, Ala40, Ser188, and Ala187 (Leu, Leu, Pro, and Leu respectively, in glycolytic Bacillus stearothermophilus GAPDH sequence) are of primary importance for the dual cofactor specificity of photosynthetic GAPDH. These modifications seem to trace the minimum evolutionary route for a primitive NAD-specific GAPDH to be converted into the NADP-preferring enzyme of oxygenic photosynthetic organisms.     

Serum levels of tartrate-resistant acid phosphatase-5b in breast cancer patients treated with pamidronate

A novel immunoassay specific for the osteoclast-produced TRAP isoform 5b has been developed recently. By means of this assay we studied the usefulness of serum TRAP-5b in monitoring the response to palliative treatment with pamidronate in breast cancer patients with bone metastases. We correlated serum TRAP-5b levels with pain intensity and intake of analgesics to assess the possible utility of the marker in identifying patients who could benefit from pamidronate treatment. Twenty-eight advanced breast cancer patients with bone metastases entered the study. Patients were treated according to the following schedule: two two-week cycles of 60 mg/week pamidronate IV, with a three-week interval in between (six infusions over seven weeks), followed by one infusion every three weeks for a total of 24 infusions over a treatment period of 61 weeks. Blood samples were taken before the start of treatment and before each infusion during two treatment cycles. To measure serum TRAP levels we employed the new immunoassay kit BoneTRAP produced by Suomen Bioanalytiikka Oy (SBA), Oulu, Finland. In order to assess the usefulness of this marker in evaluating the response to pamidronate treatment we divided patients into two groups (group A, worsened; group B, improved) with respect to pain trend and analgesic intake. Our results did not show any statistically significant difference in baseline serum TRAP levels in the two groups. However, one week after the first pamidronate infusion TRAP-5b serum levels decreased by 39% and 18% in groups A and B, respectively (p=0.01); these levels persisted throughout the treatment period. In conclusion, a decrease in TRAP-5b serum levels may reflect the pharmacological activity of pamidronate and seems to predict pain relief and a reduction in analgesic consumption.     

Metabolic modulation induced by chronic hypoxia in rats using a comparative proteomic analysis of skeletal muscle tissue

Hypoxia-induced changes of rat skeletal muscle were investigated by two-dimensional difference in-gel electrophoresis (2D-DIGE) and mass spectrometry. The results indicated that proteins involved in the TCA cycle, ATP production, and electron transport are down-regulated, whereas glycolytic enzymes and deaminases involved in ATP and AMP production were up-regulated. Up-regulation of the hypoxia markers hypoxia inducible factor 1 (HIF-1alpha) and pyruvate dehydrogenase kinase 1 (PDK1) was also observed, suggesting that in vivo adaptation to hypoxia requires an active metabolic switch. The kinase protein, mammalian target of rapamycin (mTOR), which has been implicated in the regulation of protein synthesis in hypoxia, appears unchanged, suggesting that its activity, in this system, is not controlled by oxygen partial pressure.