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Experimental radioimmunotherapy and effector mechanisms
Umeå University, Faculty of Medicine, Clinical Microbiology, Immunology/Immunchemistry. Umeå University, Faculty of Medicine, Radiation Sciences, Diagnostic Radiology. Umeå University, Faculty of Medicine, Radiation Sciences, Radiation Physics.
2006 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Radioimmunotherapy is becoming important as a new therapeutic strategy for treatment of tumour diseases. Lately monoclonal antibodies tagged with radionuclides have demonstrated encouraging results in treatment of hematological malignancies. The progress in treatment of solid tumours using radioimmunotherapy, however, has been slow. New strategies to improve the treatment response need to be evaluated. Such new strategies include the combination of radioimmunotherapy with other treatment modalities but also elucidation and exploration of the death effector mechanisms involved in tumour eradication.

As the combination of radioimmunotherapy and radiotherapy provides several potential synergistic effects, we started out by optimising a treatment schedule to detect benefits combining these treatment modalities. An anti-cytokeratin antibody labelled with 125I administered before, after, or simultaneously with radiotherapy, indicated that the highest dose to the tumour was delivered when radiotherapy was given prior to the antibody administration. The optimised treatment schedule was then applied therapeutically in an experimental study on HeLa Hep2 tumour bearing nude mice given radiotherapy prior to administration of 131I-labelled monoclonal antibodies. Combining these treatment regimes enhanced the effect of either of the treatment modalities given alone, and a significant reduction in tumour volumes could be demonstrated. This treatment caused a dramatic change in tumour morphology, with increased amounts of connective tissue, giant cells and cysts. Furthermore cellular alterations like heterogeneity of nuclear and cytoplasmic size and shape were observed, and at least a fraction of the tumour cells presented some characteristics of apoptosis.

The induced sequential events in Hela Hep2 cells exposed to 2.5-10 Gy of ionizing radiation were studied further, with special emphasis on cell cycle arrest, mitotic aberrations and finally cell death. Following radiation HeLa Hep2 cells initiated a transient G2/M arrest trying to repair cellular damage. This arrest was followed by a sequence of disturbed mitoses with anaphase bridges, lagging chromosomal material, hyperamplification of centrosomes and multipolar mitotic spindles. These mitotic disturbances produced multinuclear polyploid cells and cells with multiple micronuclei, cells that were destined to die via mitotic catastrophes and delayed apoptosis.

Induction of apoptosis in HeLa Hep2 cells following radiation doses and dose-rates equivalent to those delivered at radioimmunotherapy was concurrently studied in vitro. Significant induction of apoptosis was obtained and found to be induced relatively slowly, peaking 72-168 hours post irradiation. Caspases from the intrinsic pathway as well as the extrinsic pathway were found to be activated in response to ionizing radiation. Furthermore caspase-2, which has recently been acknowledged for its role as an initiator caspase was found to be activated following radiation and seems to play an important role in this delayed apoptosis.

Place, publisher, year, edition, pages
Umeå: Klinisk mikrobiologi , 2006. , 72 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1002
Keyword [en]
radioimmunotherapy, apoptosis, mitotic catastrophe, caspases, cell cycle disturbances, combination treatment.
National Category
Immunology
Identifiers
URN: urn:nbn:se:umu:diva-673ISBN: 91-7264-013-8 (print)OAI: oai:DiVA.org:umu-673DiVA: diva2:144206
Public defence
2006-02-10, A 103, byggnad 6A, våning 1, 90185 NUS, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2006-01-19 Created: 2006-01-19 Last updated: 2009-10-07Bibliographically approved
List of papers
1. The combination of external beam radiotherapy and experimental radioimmunotargeting with a monoclonal anticytokeratin antibody
Open this publication in new window or tab >>The combination of external beam radiotherapy and experimental radioimmunotargeting with a monoclonal anticytokeratin antibody
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2002 (English)In: Cancer, ISSN 0008-543X, Vol. 94, no S4, 1314-1319 p.Article in journal (Refereed) Published
Identifiers
urn:nbn:se:umu:diva-4919 (URN)10.1002/cncr.10302 (DOI)11877762 (PubMedID)
Available from: 2006-01-19 Created: 2006-01-19 Last updated: 2009-12-04Bibliographically approved
2. Combined low dose radio- and radioimmunotherapy of experimental HeLa Hep 2 tumours.
Open this publication in new window or tab >>Combined low dose radio- and radioimmunotherapy of experimental HeLa Hep 2 tumours.
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2003 (English)In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, Vol. 30, no 6, 895-906 p.Article in journal (Refereed) Published
Abstract [en]

Radiation therapy of malignant tumours can be delivered by external beam radiation (RT) or radioimmunotherapy (RIT), using nuclides attached to monoclonal antibodies (mAbs). These treatment modalities have now been combined in order to investigate putative therapeutic advantages and elucidate the biological responses involved. Nude mice were transplanted subcutaneously on the back with human HeLa Hep2 tumour cells. RT (3x5 Gy) and/or 100 microg (131)I-labelled mAb H7, against placental alkaline phosphatase, or (131)I-labelled mAb TS1, against cytokeratin, was administered separately or in combination (specific activity of 120-200 MBq/mg antibody). Significant tumour growth retardation was observed both with RT alone and with RIT alone. Combining these regimens enhanced the therapeutic effects further, and a significant reduction in tumour volume could be demonstrated. The tumours were subjected to extensive histochemical and immunohistochemical investigations in order to elucidate changes in biology and histology within them. The following stainings were used: haematoxylin-eosin (morphology), Ki67 (proliferation), M30 (apoptosis), TUNEL (apoptosis) and endoglin (vascularisation). Tumours in the control group grew fast, with an average tumour doubling time of 9 days. These tumours contained large viable tumour cell masses displaying vast proliferation zones of Ki67-positive tumour cells, as well as necrotic regions and small amounts of connective tissue. Apoptotic cells could be identified both with M30 and TUNEL staining. When RT was applied, the growth rate was significantly reduced (doubling time 19 days) and typical alterations in morphology were seen, with a relative increase in connective tissue and a decrease in necrotic regions. Apoptotic cells were identified and a decrease in cell density was also observed. When RIT alone was applied, the growth parameters indicated a longer lasting growth reduction, especially when TS1 was used separately or in combination with H7. The histological appearances of these tumours were somewhat different from the RT-treated tumours, with a larger portion of intratumoural cysts. These tumours also presented a reduced tumour cell density. Dramatic effects were observed when RT was combined with RIT, with a pronounced growth reduction seen in all combination treatment groups. Pronounced tumour volume reduction was also evident in both the RT + RIT ((131)I-TS1) group and RT + RIT ((131)I-TS1/(131)I-H7) group, and in some animals no tumour remained at all. The morphology of the tumour remnants at day 22 was chaotic with a drastically changed histology, with presence of abundant cysts, low fractions of Ki67-positive cells, reduction in cell density, increased amounts of connective tissue and a decrease in necrotic regions. Again, apoptotic cells could be identified, scattered throughout the viable regions. Combining RT and RIT seems to generate an efficient treatment with convincing and long-lasting tumour growth inhibition, which is reflected in a highly aberrant histology within the tumour. Results obtained in this study indicate that both necrosis and apoptosis may be involved in the process leading to this efficient therapy of epithelially derived tumours.

Identifiers
urn:nbn:se:umu:diva-4920 (URN)10.1007/s00259-003-1177-2 (DOI)12721768 (PubMedID)
Available from: 2006-01-19 Created: 2006-01-19 Last updated: 2009-12-04Bibliographically approved
3. Cell cycle disturbances and mitotic catastrophes in HeLa Hep2 cells following 2.5 to 10 Gy of ionizing radiation.
Open this publication in new window or tab >>Cell cycle disturbances and mitotic catastrophes in HeLa Hep2 cells following 2.5 to 10 Gy of ionizing radiation.
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2007 (English)In: Clin Cancer Res, ISSN 1078-0432, Vol. 13, no 18 Pt 2, 5501s-5508s p.Article in journal (Refereed) Published
Abstract [en]

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 alpha-tubulin and gamma-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.

Identifiers
urn:nbn:se:umu:diva-17688 (URN)10.1158/1078-0432.CCR-07-0980 (DOI)17875782 (PubMedID)
Available from: 2007-11-15 Created: 2007-11-15 Last updated: 2009-10-07Bibliographically approved
4. Apoptosis in HeLa Hep2 cells is induced by low-dose, low-dose-rate radiation.
Open this publication in new window or tab >>Apoptosis in HeLa Hep2 cells is induced by low-dose, low-dose-rate radiation.
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2002 (English)In: Radiation Research, ISSN 0033-7587, Vol. 158, no 5, 634-640 p.Article in journal (Refereed) Published
Identifiers
urn:nbn:se:umu:diva-4922 (URN)12385641 (PubMedID)
Available from: 2006-01-19 Created: 2006-01-19 Last updated: 2010-06-11Bibliographically approved
5. Apoptotic signalling pathways induced in HeLa Hep2 cells following 5 Gy of ionizing radiation.
Open this publication in new window or tab >>Apoptotic signalling pathways induced in HeLa Hep2 cells following 5 Gy of ionizing radiation.
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Manuscript (Other academic)
Identifiers
urn:nbn:se:umu:diva-4923 (URN)
Available from: 2006-01-19 Created: 2006-01-19 Last updated: 2010-01-13Bibliographically approved

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