Torgny Stigbrand

Radiation-induced cell death mechanisms

The main goal when treating malignancies with radiation therapy is to deprive tumor cells of their reproductive potential. One approach to achieve this is by inducing tumor cell apoptosis. Accumulating evidences suggest that induction of apoptosis alone is insufficient to account for the therapeutic effect of radiotherapy. It has become obvious in the last few years that inhibition of the proliferative capacity of malignant cells following irradiation, especially with solid tumors, can occur via alternative cell death modalities or permanent cell cycle arrests, i.e., senescence. In this review, apoptosis and mitotic catastrophe, the two major cell deaths induced by radiation, are described and dissected in terms of activating mechanisms. Furthermore, treatment-induced senescence and its relevance for the outcome of radiotherapy of cancer will be discussed. The importance of p53 for the induction and execution of these different types of cell deaths is highlighted. The efficiency of radiotherapy and radioimmunotherapy has much to gain by understanding the cell death mechanisms that are induced in tumor cells following irradiation. Strategies to use specific inhibitors that will manipulate key molecules in these pathways in combination with radiation might potentiate therapy and enhance tumor cell kill.

Neurofilament and glial fibrillary acidic protein in multiple sclerosis

Objective: To evaluate levels of neurofilament light (NFL) and glial fibrillary acidic protein (GFAP) in CSF from patients with multiple sclerosis (MS) in relation to clinical progress of the disease. Methods: CSF levels of NFL and GFAP were determined by sensitive ELISAs in 99 patients with different subtypes of MS, classified in terms of “ongoing relapse” or “clinically stable disease,” and 25 control subjects. Levels were compared with paraclinical data such as immunoglobulin G index and inflammatory cell count in the CSF, and the levels were related to Expanded Disability Status Scale score and progression index at clinical follow-up evaluations later in the disease course. Results: NFL and GFAP levels were elevated in MS patients as compared with control subjects (p < 0.001). The NFL levels were higher at relapses, whereas GFAP levels were unaffected. High NFL levels correlated with progression in patients with an active relapse (r = 0.49; p < 0.01) and in clinically stable patients (r = 0.29; p < 0.05). GFAP correlated to progression in the total patient cohort (r = 0.24; p < 0.05). Moreover, a strong correlation between NFL levels and inflammatory cell counts was evident in the group of patients with an ongoing relapse (r = 0.52; p = 0.001). Conclusions: CSF levels of neurofilament light and glial fibrillary acidic protein may have prognostic value in multiple sclerosis.

Tumour therapy with radionuclides: assessment of progress and problems

Radionuclide therapy is a promising modality for treatment of tumours of haematopoietic origin while the success for treatment of solid tumours so far has been limited. The authors consider radionuclide therapy mainly as a method to eradicate disseminated tumour cells and small metastases while bulky tumours and large metastases have to be treated surgically or by external radiation therapy. The promising therapeutic results for haematological tumours give hope that radionuclide therapy will have a breakthrough also for treatment of disseminated cells from solid tumours. New knowledge related to this is continuously emerging since new molecular target structures are being characterised and the knowledge on pharmacokinetics and cellular processing of different types of targeting agents increases. There is also improved understanding of the factors of importance for the choice of appropriate radionuclides with respect to their decay properties and the therapeutic applications. Furthermore, new methods to modify the uptake of radionuclides in tumour cells and normal tissues are emerging. However, we still need improvements regarding dosimetry and treatment planning as well as an increased knowledge about the tolerance doses for normal tissues and the radiobiological effects on tumour cells. This is especially important in targeted radionuclide therapy where the dose rates often are lower than 1Gy/h.

Epitope Specificity of 30 Monoclonal Antibodies against Cytokeratin Antigens: The ISOBM TD5-1 Workshop

The epitope specificities of 30 monoclonal antibodies (MAbs) against the most common human cytokeratins, i.e., Nos. 8, 18, and 19, in epithelial cells were investigated in the ISOBM TD-5 Workshop. Seven research groups from universities or companies participated independently in the evaluation of the antibody specificities. The complex assembly of cytokeratins in vivo, with obligatory heterologous dimeric combinations of different cytokeratins from each of the two major groups, comprising together more than 20 different individual cytokeratins, made analysis of the antibody reactivity patterns with isolated single cytokeratins necessary. The concordance of the evaluations was striking and independent of the technologies used. As antigens purified individual cytokeratins, chemically degraded purified cytokeratins, recombinant intact and truncated cytokeratins, as well as specific synthesized shorter peptides were used. In order to elucidate the epitope specificity, reactivity patterns in ELISA assays and immunoblots with partial enzymatic degradation of the antigens were performed. Competitive cross-inhibition experiments between antibodies using antigens and antibodies in all possible combinations were performed with radioimmunometric assays, BIAcore, and ELISA technology.All 30 antibodies could convincingly be classified with regard to target cytokeratin. One MAb (192) had to be deleted due to dual specificities in both isotype and epitope specificity against its target. Six antibodies bound selectively to cytokeratin 8, 14 to cytokeratin 18, and 10 to cytokeratin 19, as demonstrated by using native, recombinant, and synthesized antigens. The immunodominant part of the molecule for all three types of cytokeratins was located in the region of amino acid (aa) 270–400. Out of the six MAbs reactive with cytokeratin 8, four MAbs, i.e., 178, 199, 202, and 206, were reactive with a sequence in the interval aa 340–365, and MAb 191 reacted with a closely related epitope. The remaining antibody, 192, presented dual specificities. At least two closely related major immunogenic epitopes could be identified in cytokeratin 8. In cytokeratin 18 four distinct epitopes could be documented, again with the dominating sequence region 270–429 as target for 10 (181, 184, 186, 188, 189, 190, 193, 196, 198, and 200) out of 14 antibodies. Since MAb 193 is known to react with the M3 epitope, aa 322–342 in cytokeratin 18, this entire group is reactive in the region close to the charge shift, in the middle of the rod 2B region, as shown by competitive binding. The remaining four anticytokeratin 18 antibodies (180, 185, 203, and 205) displayed unique, noncompetitive binding to this filament. Cytokeratin 19, reactive with altogether ten antibodies, displayed two major epitopes, all of them also within the large immunodominant region. MAbs 179, 195, 197, and 204 were reactive with the peptides aa 311–335 also known as the KS 19.1 epitope, and MAbs 182, 183, 187, 194, and 201 bound to peptide aa 346–367, known as the BM 19.21 epitope. One antibody, 231, was selectively reactive with aa 356–370 in cytokeratin 19. A complex pattern of binding specificities comprising at least ten different, noncompetitive epitopes, mainly situated in the rod portion, 2A and 2B, situated close to the charge shift in the rod of all three cytokeratins was documented. Out of the 29 classifiable antibodies, altogether 22 were reactive in this very short region, i.e., from aa 311 to 370 in all cytokeratin filaments. The remaining seven antibodies displayed unique binding properties. The implications of the findings are of significance both for immunohistochemistry and for assaying circulating heterodimeric, partially degraded complexes in patients’ blood for tumor marker evaluation.

Inhibition of PHA-induced lymphocyte stimulation by the pregnancy zone protein

Mother and child are genetically different and the fetus could be looked upon as an allograft. The reason why this allograft is accepted is unknown. A physiological barrier between mother and fetus [ 1 ], and the presence of blocking antibodies [2] and hormones [3] have been suggested. A depression of the maternal cellmediated immune reactivity [4-6] is another explanation supported by the report of Kasakura [7] that maternal plasma during pregnancy suppressed the mixed leucocyte reaction. The concentration in plasma of many proteins is altered during pregnancy but some proteins are produced in extensively increased amounts. The pregnancy zone protein (PZ) [8-16] is an a2-globulin which is absent or present in only small amounts in the sera of non-pregnant women. During pregnancy the serum level of this globulin is increased in most women from 0 4 rag/100 ml to values between 50-200 mg/100 ml. The maximum is generally reached in the third trimester and after delivery there is a rapid decrease to normal levels [ 17]. In women with early spontaneous abortion PZ is found rarely or in small amounts [18]. The PZ protein has been isolated and found to have a mol. wt. of 326 000 [19]. In the present study PZ was found to inhibit the phytohaemagglutinin (PHA)-induced lymphocyte stimulation, believed to be an in vitro correlate of cellmediated immunity. 2. Materials and methods

S-100β has a neuronal localisation in the rat hindbrain revealed by an antigen retrieval method

The localisation of S-100 in mammalian CNS neurons has been under debate for more than two decades. We address the question with two polyclonal and two new monoclonal antibodies. The specificity and the distribution in rat brain is based on an antigen retrieval method. We present evidence that aldehyde fixatives mask S-100 beta in neurons, and that the immunoreactivity is retrieved after trypsinisation. Neuronal S-100 beta is also detected in unfixed and ethanol fixed sections. The neuronal immunoreactivity is partly solubilised from unfixed tissue sections with 2.5 mM EDTA and is completely extracted with 2.5 mM EDTA and 1% Triton X-100. Most of the glial S-100 beta is washed out from unfixed tissue sections with saline. S-100 beta has distinct distribution in neurons of the hindbrain, i.e., the brainstem and cerebellum, but is not observed in the forebrain. One of the monoclonal antibodies immunostained neither neurons nor glia when it had been absorbed with S-100 crosslinked to nitrocellulose membranes. The distribution of neuronal S-100 beta differed from that of other neuronal calcium binding proteins, such as calbindin and parvalbumin. It was confined mainly to cholinergic neurons of the hindbrain. The presence of S-100 beta in distinct neuronal populations may indicate neurotrophic effects of S-100 beta. The notion is supported by the capability of S-100 to cause neurite outgrowth in vitro.

Immunolocalization of stratum corneum chymotryptic enzyme in human skin and oral epithelium with monoclonal antibodies: evidence of a proteinase specifically expressed in keratinizing squamous epithelia.

Stratum corneum chymotryptic enzyme (SCCE) is a recently discovered serine proteinase, which has been purified from human plantar stratum corneum. Evidence has been presented that it may play a role in the terminal stages of epidermal turnover, especially in desquamation. Two mouse monoclonal antibodies (MAb) were raised, TE4b and TE9b, that reacted specifically with SCCE in immunoprecipitation, immunoblotting, and gel-exclusion chromatography. When used in immunohistochemical experiments with the peroxidase-anti-peroxidase method, both MAb detected an antigen located in high suprabasal keratinocytes of the epidermis in normal human skin and at the vermilion border of the lip, with maximal staining of the stratum granulosum. In the hair follicles the MAb reacted with the inner root sheet only. In human oral mucosa the MAb stained the high suprabasal epithelial cells of the hard palate. This is a site where the epithelium forms an orthokeratotic stratum corneum. There was no specific staining of the epithelium of the lip mucosa or the buccal mucosa, where the epithelium does not form a stratum corneum under non-pathological conditions. A correlation therefore seems to exist between the presence of SCCE in high suprabasal cells and the ability of the epithelium to form an orthokeratotic cornified layer. We suggest that SCCE is specifically expressed in keratinizing squamous epithelia and that its expression may be part of the terminal differentiation program of this type of epithelium. These results also give further support to the idea that SCCE may play a role in the turnover and/or formation of the stratum corneum.

Immobilized metal ion affinity partitioning, a method combining metal-protein interaction and partitioning of proteins in aqueous two-phase systems.

Immobilized metal ions were used for the affinity extraction of proteins in aqueous two-phase systems composed of polyethylene glycol (PEG) and dextran or PEG and salt. Soluble chelating polymers were prepared by covalent attachment of metal-chelating groups to PEG. The effect on the partitioning of proteins of such chelating PEG derivatives coordinated with different metal ions is demonstrated. The proteins studied were alpha 2-macroglobulin, tissue plasminogen activator, superoxide dismutase and monoclonal antibodies. The results indicate that immobilized metal ion affinity partitioning provides excellent potential for the extraction of proteins.

Apoptosis induced by low-dose and low-dose-rate radiation

It has been claimed that external radiation, as a treatment modality for malignant diseases, partly induces apoptosis. It is not known, however, whether therapeutic low‐dose and low‐dose‐rate radiation are able to induce apoptosis.

Cell Cycle Disturbances and Mitotic Catastrophes in HeLa Hep2 Cells following 2.5 to 10 Gy of Ionizing Radiation

Purpose: Experimental radioimmunotherapy delivering absorbed doses of 2.5 to 10 Gy has been shown to cause growth retardation of tumors. The purpose of this study was to elucidate the sequential molecular and cellular events occurring in HeLa Hep2 cells exposed to such doses. Methods: Dose-response curves, activation of cell cycle checkpoints, and mitotic behavior were investigated in HeLa Hep2 cells following 2.5- to 10-Gy irradiation by carrying out 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, Western blots, fluorescence-activated cell sorting analysis, and immunofluorescence stainings. Terminal deoxyribonucleotidyl transferase–mediated dUTP nick end labeling staining was used to detect apoptosis. Results: A G2-M arrest was shown by fluorescence-activated cell sorting analysis. p53 and p21 were found to be up-regulated but were not immediately related to the arrest. The G2-M arrest was transient and the cells reentered the cell cycle still containing unrepaired cellular damage. This premature entry caused an increase of anaphase bridges, lagging chromosomal material, and multipolar mitotic spindles as visualized by propidium iodide staining and immunofluorescence staining with α-tubulin and γ-tubulin antibodies. Furthermore, a dose-dependent significant increase in centrosome numbers from 12.6 ± 6.6% to 67 ± 5.3% was identified as well as a dose-dependent increase of polyploid cells from 2.8 ± 1.3% to 17.6 ± 2.1% with the highest absorbed dose of 10 Gy. These disturbances caused the cells to progress into mitotic catastrophe and a fraction of these dying cells showed apoptotic features as displayed by terminal deoxyribonucleotidyl transferase–mediated dUTP nick end labeling staining 5 to 7 days after irradiation. Conclusion: An absorbed dose of 2.5 to 10 Gy was shown to force HeLa Hep2 cells into mitotic catastrophe and delayed apoptosis. These might be important cell death mechanisms involved in tumor growth retardation following radioimmunotherapy of solid tumors.