Erik O. Pettersen

The mechanism of photodynamic inactivation of human cells In Vitro in the presence of haematoporphyrin

The photosensitizing effect of haematoporphyrin (HP) on human cells of the established line NHIK 3025 has been studied. Fluorescence measurements show that HP is bound to these cells. Serum proteins also bind HP, and the presence of 10% human serum during incubation with HP (3 X 10(-4)M) reduces the cellular uptake of HP by 75% or more. The photosensitized inactivation is enhanced when the cells are suspended in D2O-buffer during irradiation. This indicates that singlet oxygen is involved in the inactivation. Two findings indicate that the photoinduced damage is repairable: firstly, the fraction of cells surviving a given light dose decreases with decreasing irradiation temperature, and secondly, the survival curves have a shoulder at low exposures of light.

Taking advantage of tumor cell adaptations to hypoxia for developing new tumor markers and treatment strategies.

Cancer cells in hypoxic areas of solid tumors are to a large extent protected against the action of radiation as well as many chemotherapeutic drugs. There are, however, two different aspects of the problem caused by tumor hypoxia when cancer therapy is concerned: One is due to the chemical reactions that molecular oxygen enters into therapeutically targeted cells. This results in a direct chemical protection against therapy by the hypoxic microenvironment, which has little to do with cellular biological regulatory processes. This part of the protective effect of hypoxia has been known for more than half a century and has been studied extensively. However, in recent years there has been more focus on the other aspect of hypoxia, namely the effect of this microenvironmental condition on selecting cells with certain genetic prerequisites that are negative with respect to patient prognosis. There are adaptive mechanisms, where hypoxia induces regulatory cascades in cells resulting in a changed metabolism or changes in extracellular signaling. These processes may lead to changes in cellular intrinsic sensitivity to treatment irrespective of oxygenation and, furthermore, may also have consequences for tissue organization. Thus, the adaptive mechanisms induced by hypoxia itself may have a selective effect on cells, with a fine-tuned protection against damage and stress of many kinds. It therefore could be that the adaptive mechanisms may take advantage of for new tumor labeling/imaging and treatment strategies. One of the Achilles’ heels of hypoxia research has always been the exact measurements of tissue oxygenation as well as the control of oxygenation in biological tumor models. Thus, development of technology that can ease this control is vital in order to study mechanisms and perform drug development under relevant conditions. An integrated EU Framework project 2004–2009, termed EUROXY, demonstrates several pathways involved in transcription and translation control of the hypoxic cell phenotype and evidence of cross-talk with responses to pH and redox changes. The carbonic anhydrase isoenzyme CA IX was selected for further studies due to its expression on the surface of many types of hypoxic tumors. The effort has led to marketable culture flasks with sensors and incubation equipment, and the synthesis of new drug candidates against new molecular targets. New labeling/imaging methods for cancer diagnosing and imaging of hypoxic cancer tissue are now being tested in xenograft models and are also in early clinical testing, while new potential anti-cancer drugs are undergoing tests using xenografted tumor cancers. The present article describes the above results in individual consortium partner presentations.

Analysis of prognostic factors in stage I epithelial ovarian carcinoma : importance of degree of differentiation and deoxyribonucleic acid ploidy in predicting relapse

OBJECTIVES Our purpose was to identify prognostic factors in stage I epithelial invasive ovarian carcinoma. STUDY DESIGN The traditional clinical and pathologic prognostic variables and deoxyribonucleic acid ploidy were analyzed in a group of 290 patients. RESULTS A multivariate analysis identified degree of differentiation as the most powerful prognostic indicator of disease-free survival, followed by deoxyribonucleic acid ploidy and, finally, International Federation of Gynecology and Obstetrics (1986) stage. Tumors with clear cell carcinoma elements were not graded, and in this subgroup International Federation of Gynecology and Obstetrics (1986) stage was the most important prognostic characteristic. When the effects of the three most important factors were accounted for in graded tumors, then none of the following were prognostic: histologic type, dense adhesion, extracapsular growth, ascites, rupture during surgery, International Federation of Gynecology and Obstetrics (1973) stage, size of tumor, and age and type of adjuvant treatment. None of 77 patients with well differentiated deoxyribonucleic acid diploid tumors had relapses. CONCLUSION Deoxyribonucleic acid ploidy is an important new independent prognostic factor in stage I ovarian carcinoma.

Cell inactivation and cell cycle inhibition as induced by extreme hypoxia: the possible role of cell cycle arrest as a protection against hypoxia‐induced lethal damage

Abstract. Cycling mammalian cells that are rendered extremely hypoxic 4 ppm O2 tend to accumulate in a pre‐DNA‐synthesis stage. It is not clear whether or not this is the result of an active regulation by the cells. In the present study we have rendered cells, synchronized by mitotic selection, extremely hypoxic over a relatively long period of time (up to 48 h). We have recorded cell cycle progression during hypoxia as well as cell inactivation depending on where in the cell cycle the cells were located when the hypoxic treatment was started. Three main conclusions are drawn: 1 the cell cycle arrest in late‐G1 is complete even during a long‐lasting (24 h) hypoxic treatment; 2 while cells in early‐ and mid‐S are completely arrested and quickly inactivated under hypoxic conditions, cells in late‐S, G2 and mitosis are able to continue cell cycle progression and divide; 3 whether the cells are located in G2, mitosis or early‐G1 at the onset of hypoxia, they were able to survive relatively long‐lasting hypoxic treatment. The present results are in favour of the view that the pre‐DNA‐synthetic arrest induced by extreme hypoxia may function to rescue the cells from severely damaging effects that would appear if the cells were able to initiate DNA synthesis.

Randomized study on adjuvant chemotherapy in stage I high-risk ovarian cancer with evaluation of DNA-ploidy as prognostic instrument

Summary Purpose: Adjuvant chemotherapy versus observation and chemotherapy at progression was evaluated in 162 patients in a prospective randomized multicenter study. We also evaluated DNA-measurements as an additional prognostic factor. Patients and methods: Patients received adjuvant carboplatin AUC 7 every 28 days for six courses (n - 81) or no adjuvant treatment (n = 81). Eligibility included surgically staged and treated patients with FIGO stage I disease, grade 1 aneuploid or grade 2 or 3 non-clear cell carcinomas or clear cell carcinomas. Disease-free (DFS) and disease-specific (DSS) survival were end-points. Results: Median follow-up time was 46 months and progression was observed in 20 patients in the treatment group and 19 in the control group. Estimated five-year DFS and DSS were 70% and 86% in the treatment group and 71% and 85% in the control group. The hazard ratio was 0.98 (95% confidence interval (95% CI): 0.52-1.83) regarding DFS and 0.94 (95% CI: 0.37-2.36) regarding DSS. No significant differences in DFS or DSS could be seen when the log-rank test was stratified for prognostic variables. Therefore, data from both groups were pooled for the analysis of prognostic factors. DNAploidy (P = 0.003), extracapsular growth (P = 0.005), tumor rupture (P = 0.04), and WHO histologic grade (P = 0.04) were significant independent prognostic factors for DFS with P < 0.0001 for the model in the multivariate Cox analysis. FIGO substage (P = 0.01), DNA ploidy (P < 0.05), and histologic grade (P = 0.05) were prognostic for DSS with a P-value for the model < 0.0001. Conclusions: Due to the small number of patients the study was inconclusive as regards the question of adjuvant chemotherapy. The survival curves were superimposable, but with wide confidence intervals. DNA-ploidy adds objective independent prognostic information regarding both DFS and DSS in early ovarian cancer.

Histologic subtype has minor importance for overall survival in patients with adenocarcinoma of the uterine cervix: A population-based study of prognostic factors in 505 patients with nonsquamous cell carcinomas of the cervix

The incidence of adenocarcinoma of the uterine cervix is increasing. For better prognostic information, the authors studied all nonsquamous cell carcinomas (non‐SCCs) in the Norwegian population over a total of 15 years.

Pericellular oxygen depletion during ordinary tissue culturing, measured with oxygen microsensors

Abstract.  Recent research has found important differences in oxygen tension in proximity to certain mammalian cells when grown in culture. Oxygen has a low diffusion rate through cell culture media, thus, as a result of normal respiration, a decrease in oxygen tension develops close to the cells. Therefore, for the purpose of standardization and optimization, it is important to monitor pericellular oxygen tension and cell oxygen consumption. Here, we describe an integrated oxygen microsensor and recording system that allows measurement of oxygen concentration profiles in vertical transects through a 1.6‐mm deep, stagnant, medium layer covering a cell culture. The measurement set‐up reveals that, when confluent, a conventional culture of adherent cells, although exposed to the constant oxygen tension of ambient air, may experience pericellular oxygen tensions below the level required to sustain full oxidative metabolism. Depletions reported are even more prominent and potentially aggravating when the cell culture is incubated at reduced oxygen tensions (down to around 4% oxygen). Our results demonstrate that, if the pericellular oxygen tension is not measured, it is impossible to relate in vitro culture results (for example, gene expression to the oxygen tension experienced by the cell), as this concentration may deviate very substantially from the oxygen concentration recorded in the gas phase.

DNA ploidy; the most important prognostic factor in patients with borderline tumors of the ovary

The prognostic significance of DNA ploidy in relation to clinical and histopathologic factors was evaluated in a retrospective study of 370 patients treated at the Norwegian Radium Hospital from 1970 to 1982 with complete follow-up of median 149 months. Evaluable flow cytometric DNA histograms from paraffin-embedded tissue from the primary tumor were obtained in 321 cases, 293 (91%) were diploid and 28 (9%) were aneuploid. Aneuploidy was associated with older age, more advanced disease and non-serous histologic types. By multivariate analysis the only parameters with prognostic significance for corrected survival (death from disease) were ploidy, stage, histologic type and age. The patients with aneuploid tumors had a 19-fold increased risk of dying of disease compared with patients with diploid tumors. In tumor-free operated patients the extent of surgery had no influence on survival, neither had postoperative treatment. Using the prognostic factors the patients could be divided into risk groups. The large group of patients with diploid stage I tumors belonged to the low risk group. Fertility-saving operations can be offered to patients with diploid stage IA tumors, all others should have bilateral salpingo-oophorectomy and omentectomy with or without hysterectomy. Patients with diploid stage I tumors should not receive adjuvant treatment. The value of adjuvant chemotherapy in the high risk group needs further investigation.

The Prognostic Significance of ‘Stage, Tumor Size, Cellular Atypia and DNA Ploidy in Uterine Leiomyosarcoma

To analyze the significance of DNA ploidy in uterine leiomyosarcoma, the traditional clinical and histopathological prognostic variables and DNA ploidy were studied in 70 patients with histologically verified uterine leiomyosarcoma. Evaluable flow cytometric DNA histograms from paraffin-embedded tissue from the tumor were obtained in 58 patients. In univariate analysis tumor diameter, FIGO stage and presence of residual disease after primary surgery were highly significant (p < 0.001) and also DNA ploidy (p = 0.043), age (p = 0.017), and menopause status (p = 0.028) obtained significance. Cellular atypia was almost significant (p = 0.056), while mitotic count, malignancy grade and vessel invasion were not. In Coxs multivariate analysis, FIGO-stage was found to be the most important prognostic factor (p < 0.001), followed by cellular atypia (p = 0.007) and tumor diameter (p = 0.016). DNA ploidy did not obtain significance when categorized as diploid/non-diploid. Patients with tumors with multiple aneuploid cell populations had a very poor prognosis. When categorized as multiple aneuploidy versus all other ploidy groups, DNA ploidy obtained marginal significance in multivariate analysis (p = 0.054). Tumor diameter, stage and cellular atypia are important prognostic parameters in uterine leiomyosarcomas.

Spermatocytic seminoma as compared to classical seminoma: An immunohistochemical and DNA flow cytometric study

Based on immunohistochemistry (IHC) and DNA ploidy, different paths of carcinogenesis have been suggested for spermatocytic seminoma (SS) and classical seminoma (CS). The present study extends current knowledge on the above parameters. Method. Seventeen SSs and twenty‐two CSs were assessed by IHC for placental‐like alkaline phosphatase (PLAP), c‐kit, cytokeratin and adhesion carbohydrate molecyles. All SSs and 11 CSs were also analysed for DNA ploidy. Results. All CSs, but none of the SSs, were positive for PLAP. C‐kit positivity was found in 7 of 17 SSs and in all CSs. The other IHC parameters were similarly distributed among the evaluated SSs and CSs. Fourteen SSs were diploid or polyploid, and three were aneuploid. All CSs were aneuploid. Conclusion. The new observation of c‐kit positivity in about 40% of SSs suggests that at least some of the SSs originate from primordial cells. The predominantly diploid or polyploid DNA pattern indicates that SSs follow a pathogenetic pathway which is most probably different from that of CSs.