Transglutaminase crosslinking and structural studies of the human small proline rich 3 protein.

The cell envelope (CE) is a vital structure for barrier function in terminally differentiated dead stratified squamous epithelia. It is assembled by transglutaminase (TGase) cross-linking of several proteins, including SPR3 in certain specialized epithelia normally subjected to mechanical trauma. We have expressed recombinant human SPR3 in order to study its cross-linking properties. It serves as a complete substrate for, and is cross-linked at similar efficiencies by, the three enzymes (TGases 1, 2 and 3) that are widely expressed in many epithelia. Multiple adjacent glutamines (4, 5, 16, 17, 18, 19 and 167) and lysines (6, 21, 164, 166 and 168) of only head and tail domain sequences are used for cross-linking. However, each enzyme preferentially uses certain residues on the head domain. Moreover, our in vitro data suggest a defined temporal order of cross-linking of SPR3 in vivo: It is first cross-linked by TGase 3 into short intra- and inter-chain oligomers which are later further cross-linked to the CE by TGase 1. To investigate the absence of cross-linking in the central domain (e.g. lysine in position 2 of each of the 16 repeats) we performed structural studies on recombinant SPR3 and on a synthetic peptide containing three repeats of the central domain. 2D H-1 NMR spectroscopy, TOCSY and ROESY, shows strong and medium intensity NOEs connectivities along the amino acid sequence with one weak long range NOE contact between Thr and Cys of subsequent repeats. Distance geometry computation on the basis of intensities of NOEs found generated 50 compatible structures grouped in three main families differing by the number of H-bonds. These measurements were repeated at different concentrations of trifluoroethanol (TFE)-water mixture, an alpha-helical promoting solvent, in order to check the stability of the conformations determined; no changes were observed up to 50% TFE in solution. Also temperature changes did not produce any variation in the ROESY spectrum in the same condition as above. The NMR and circular dichroism data strongly indicate the presence of an ordered (not alpha-helix nor beta-sheet) highly flexible structure in the eight amino acids repetitive units of SPR3, confirming the prediction of one possible beta-turn per each repeating unit. Thus, biochemical and biophysical data, strongly support SPR3 to function as a flexible cross-bridging protein to provide tensile strength or rigidity to the CE of the stratified squamous epithelia in which it is expressed.

Antitumor activity of combined treatment of human cancer cells with ionizing radiation and anti-epidermal growth factor receptor monoclonal antibody C225 plus type I protein kinase A antisense oligonucleotide.

Recent studies have suggested that selective inhibition of mitogenic pathways may improve the antitumor activity of ionizing radiation. The epidermal growth factor receptor (EGFR) is overexpressed and is involved in autocrine growth control in the majority of human carcinomas. Protein kinase A type I (PKAI) plays a key role in neoplastic transformation and is overexpressed in cancer cells in which an EGFR autocrine pathway is activated. We used two specific inhibitors of EGFR and PKAI that are under clinical evaluation in cancer patients: C225, an anti-EGFR chimeric human-mouse monoclonal antibody (MAb); and a mixed-backbone antisense oligonucleotide targeting the PKAI RIalpha subunit (PKAI AS). We tested in human colon cancer (GEO) and ovarian cancer (OVCAR-3) cell lines the antiproliferative activity of MAb C225 and/or PKAI AS in combination with ionizing radiation. In vivo antitumor activity was evaluated in nude mice bearing established GEO xenografts. Dose-dependent inhibition of soft agar growth was observed in both cancer cell lines with ionizing radiation, C225, or PKAI AS oligonucleotide. A cooperative antiproliferative effect was obtained when cancer cells were treated with ionizing radiation followed by MAb C225 or PKAI AS oligonucleotide. This effect was observed at all doses tested in both GEO and OVCAR-3 cancer cell lines. A combination of the three treatments at the lowest doses produced an even greater effect than that observed when two modalities were combined. Treatment of mice bearing established human GEO colon cancer xenografts with radiotherapy (RT), MAb C225, or PKAI AS oligonucleotide produced dose-dependent tumor growth inhibition that was reversible upon treatment cessation. A potentiation of the antitumor activity was observed in all mice treated with RT in combination with MAb C225 or PKAI AS oligonucleotide. Long-term GEO tumor growth regression was obtained following treatment with ionizing radiation in combination with MAb C225 plus PKAI AS oligonucleotide, which produced a significant improvement in survival compared with controls (P < 0.001), the RT-treated group (P < 0.001), or the group treated with MAb C225 plus PKAI AS oligonucleotide (P < 0.001). All mice of the RT + MAb C225 + PKAI AS group were alive 26 weeks after tumor cell injection. Furthermore, 50% of mice in this group were alive and tumor-free after 35 weeks. This study provides a rationale for evaluating in cancer patients the combination of ionizing radiation and selective drugs that block EGFR and PKAI pathways.

Brachytherapy in the elderly.

A rapidly and constantly increasing aged population in the western countries poses a wide range of specific problems to oncologists. A different way to face medical issues should be sought for older patients with cancer, looking at the characteristics that are peculiar to the elderly from different points of view. Brachytherapy is an effective form of radiotherapy which, for its specific characteristics, may be a valid alternative to more complex modalities of treatment, thus allowing a better sparing of normal tissues and structures yet achieving a similar tumor control rate. This paper reviews the literature on the subject of cancer treatment in the elderly, focusing on radiotherapy and brachytherapy, to evaluate the current attitude toward this problem in the medical community and to see if it is possible to identify a patient population that will benefit from this technique.

Antagonism between catalase and ascorbic acid in control of normal and neoplastic cell multiplication.

The authors studied the effects of a treatment with ascorbic acid on in vitro multiplication of ascites tumour cells (ATP C+), of fibroblast-like cells and of hepatocytes from chick embryos, by measuring [3H]thymidine incorporation into DNA. The results obtained show that the ATP C+ cells are the most sensitive to the toxic effects of the experimental treatment, while the hepatocytes are the most resistant cell population. A treatment with catalase was able to greatly reduce the damage caused by ascorbic acid on the ATP C+ cells. It is hypothesized that ascorbic acid inhibits cell multiplication by the H2O2 formed by its oxidation and that the cells having the highest level of catalase are more resistant to its toxic effects.

Tamoxifen and somatostatin affect tumours by inducing apoptosis.

Tamoxifen is a commonly used chemotherapeutic agent in human breast cancer, although some tumours develop resistance. Somatostatin is also being introduced as an anti-tumour agent. Here we show that the action of these drugs is, at least partly, due to their induction of apoptosis. Both 50 nM somatostatin, and 60 nM tamoxifen significantly enhanced the percentage of cells undergoing apoptosis, when compared to untreated or oestrogen treated control cells. This effect was observed in SK-N-BE(2) human neuroblastoma cells and in MCF-7G human breast cancer cells but not in their drug-resistant counterpart MCF-7A which showed a very low rate of spontaneous programmed cell death. Finally, we propose a simple test of the sensitivity and resistance of individual tumours to these agents by assessing their ability to induce apoptosis in vitro as measured by flow cytometry.

Cell multiplication of an ascites tumour in the presence of superoxide dismutase and catalase.

Superoxide dismutase and catalase were demonstrated to favour the multiplication of ascites tumour cells in vitro. It is proposed that these enzymes neutralize the O2-. and H2O2 that may accumulate in the neoplastic cell and that cell damage occurs because the cellular levels of both enzymes are low.

Effects of ascorbic and dehydroascorbic acid on the multiplication of tumor ascites cells in vitro.

The effects of AA and DHA on ATP C+ cell multiplication in vitro were studied by measuring incorporation of 3H thymidine into DNA. The results obtained demonstrate that both AA and DHA have the same effects: they favor cell multiplication at low doses and inhibit it at high doses. Experiments carried out with serial doses of both these substances revealed that AA is more efficient in determining both stimulating and inhibiting effects. The lesser efficiency of DHA may be attributed to its limited stability in culture medium. Studies on the effect of high doses of AA and DHA added to the culture medium in single or fractionated doses revealed that fractionated administration is more efficient in inhibiting cell multiplication than single administration.

Bone formation markers and pain palliation in bone metastases treated with strontium-89.

In several bone disorders, including those with metastatic involvement, changes in procollagen type I C-terminal and type III N-terminal peptides are detected, as indications of altered bone metabolism. Assessment of bone turnover could play a role in the evaluation of response to Strontium-89 used as palliative treatment in symptomatic bone metastases from various primary tumors. A correlation between bone formation rate markers procollagen I and III and efficacy of ionic Strontium-89 was shown in a group of 13 patients who underwent treatment with 4 mCi of Strontium-89 for painful bone metastases: 5 from breast, 7 from prostate, and 1 from lung carcinoid cancer. Assessed as a modification of analgesic intake, pain, and ambulation, there were 6 complete remissions, 3 partial remissions, and 4 nonresponders. The duration of the response was from 2 to 11 months. Procollagen I and III levels were found to be highly abnormal in those with no benefit from Strontium-89 administration but were in the normal range or only slightly elevated in those achieving complete or partial pain control, thus correlating with the clinical response.

Putative role of antioxidant enzymes and glyoxalases in carcinogenesis.

The effects various drugs exert on antioxidant enzyme and glyoxalase activity in rat livers were studied. All drugs tested provoked a marked reduction in glutathione peroxidase and a small drop in both glyoxalase I and II activity. It is hypothesized that the substances tested support tumour development by neutralizing organic peroxides, thereby favouring the oxidation of carcinogens and, as a consequence, the formation of metabolites that trigger neoplastic transformation. The reduction in glyoxalase activity is probably attributable to the enhanced cell proliferation induced by the treatment.

The role of antioxidant enzymes in benzo(a)pyrene-induced carcinogenesis.

Chick embryo hepatocytes were cultured in the presence of benzo(a)pyrene in order to study the effects of this carcinogen on catalase, glutathione peroxidase and superoxide dismutase activity. The results demonstrate that benzo(a)pyrene is incapable of modifying the activity of these enzymes, even though it is taken up by cultured cells to form benzo(a)pyrene-DNA adducts. The effect of culturing, however, caused a marked reduction in the activity of these enzymes. The significance of these activity variations in benzo(a)pyrene in vitro carcinogenesis is discussed.