Papers by Sakari Hietanen
Clinical Cancer Research, 2018
Purpose: Homologous recombination deficiency (HRD) correlates with platinum sensitivity in patien... more Purpose: Homologous recombination deficiency (HRD) correlates with platinum sensitivity in patients with ovarian cancer, which clinically is the most useful predictor of sensitivity to PARPi. To date, there are no reliable diagnostic tools to anticipate response to platinum-based chemotherapy, thus we aimed to develop an ex vivo functional HRD detection test that could predict both platinum-sensitivity and patient eligibility to targeted drug treatments. Experimental Design: We obtained a functional HR score by quantifying homologous recombination (HR) repair after ionizing radiation-induced DNA damage in primary ovarian cancer samples (n = 32). Samples clustered in 3 categories: HR-deficient, HR-low, and HR-proficient. We analyzed the HR score association with platinum sensitivity and treatment response, platinum-free interval (PFI) and overall survival (OS), and compared it with other clinical parameters. In parallel, we performed DNA-sequencing of HR genes to assess if functional...
Acta Oncologica, 2020
Objective: Human epididymis protein 4 (HE4) is a validated, complementary biomarker to cancer ant... more Objective: Human epididymis protein 4 (HE4) is a validated, complementary biomarker to cancer antigen 125 (CA125) for high grade serous ovarian carcinoma (HGSC). Currently, there are insufficient data on the utility of longitudinal HE4 measurement during HGSC treatment and follow up. We set to provide a comprehensive analysis on the kinetics and prognostic performance of HE4 with serial measurements during HGSC treatment and follow up. Methods: This prospective study included 143 patients with advanced HGSC (ClinicalTrials.gov identifier: NCT01276574). Serum CA125 and HE4 were measured at baseline, before each cycle of chemotherapy and during follow up until first progression. Baseline biomarker values were compared to the tumor load assessed during surgery and to residual disease. Biomarker nadir values and concentrations at progression were correlated to survival. Results: The baseline HE4 concentration distinguished patients with a high tumor load from patients with a low tumor load assessed during surgery (p<.0001). The baseline CA125 level was not associated with tumor load to a similar extent (p¼.067). At progression, the HE4 level was an independent predictor of worse survival in the multivariate analysis (p¼.002). All patients that were alive 3 years postprogression had a serum HE4 concentration below 199.20 pmol/l at the 1st recurrence. Conclusion: HE4 is a feasible biomarker in the treatment monitoring and prognostic stratification of patients with HGSC. Specifically, the serum level of HE4 at first relapse was associated with the survival of patients and it may be a useful complementary tool in the selection of second line treatments. This is to the best of our knowledge the first time this finding has been reported.
Cancer Research, 2006
p53 is degraded in cervical cancer cells by the human papillomavirus E6 and can be stabilized wit... more p53 is degraded in cervical cancer cells by the human papillomavirus E6 and can be stabilized with short interfering RNA (siRNA) molecules targeting E6 mRNA. In this in vitro study, we show that E6 siRNA-induced p53 activation is transient in HeLa cervical cancer cells despite continuous suppression of E6 mRNA; activation can be sustained if the endogenous p53 antagonists COP1, MDM2, Pirh2, and c-Jun-NH 2-kinase are also targeted by siRNAs or by inhibiting the nuclear export of p53 with leptomycin B. The direct targeting of any one of these four cellular p53 antagonists had no effect on p53 activity when E6 was intact, but inhibited the fading off of E6 siRNA-induced p53 activation in nonstress conditions. The effect was additive when multiple cellular antagonists were concomitantly inhibited, indicating that all these proteins degrade p53 when E6 is inactivated. The antiproliferative effect induced by E6 silencing was enhanced when the endogenous p53 antagonists were additionally targeted. In conclusion, if human papillomavirus E6 is inhibited under nonstress conditions, the subsequent p53 activation is quickly reversed by the endogenous p53 degenerative machinery. The present results indicate that several cellular p53 antagonists must be inhibited for sustained p53 activity if E6 siRNA therapy is attempted and if no combined genotoxic therapy is applied.
Australian and New Zealand Journal of Obstetrics and Gynaecology
To evaluate morbidity and subjective outcome associated with hysterectomy either with or without ... more To evaluate morbidity and subjective outcome associated with hysterectomy either with or without pelvic or pelvic and para-aortic lymphadenectomy for gynaecological cancer. Ninety-nine patients who underwent hysterectomy with lymphadenectomy (n = 38) or simple hysterectomy (n = 61) for ovarian, endometrial and cervical cancer in Turku University Hospital, Turku, Finland, were followed-up prospectively to determine the incidence of complications during a 1-year period after operation. Subjective outcomes were assessed using two questionnaires, 6 weeks and 1 year after operation. Hospital records of the patients were reviewed up to 6 years after operation. During their hospital stay 58% of patients in the hysterectomy with lymphadenectomy group and 56% in the simple hysterectomy group experienced some type of complication. Serious complications occurred in four patients (10.5%) in the former group and in two patients (3.3%) in the latter group. In the study population overall, the inc...
PLoS ONE, 2014
Although c-Abl has increasingly emerged as a key player in the DNA damage response, its role in t... more Although c-Abl has increasingly emerged as a key player in the DNA damage response, its role in this context is far from clear. We studied the effect of inhibition of c-Abl kinase activity by imatinib with chemotherapy drugs and found a striking difference in cell survival after combined mitoxantrone (MX) and imatinib treatment compared to a panel of other chemotherapy drugs. The combinatory treatment induced apoptosis in HeLa cells and other cancer cell lines but not in primary fibroblasts. The difference in MX and doxorubicin was related to significant augmentation of DNA damage. Transcriptionally active p53 accumulated in cells in which human papillomavirus E6 normally degrades p53. The combination treatment resulted in caspase activation and apoptosis, but this effect did not depend on either p53 or p73 activity. Despite increased p53 activity, the cells arrested in G2 phase became defective in this checkpoint, allowing cell cycle progression. The effect after MX treatment depended partially on c-Abl: Short interfering RNA knockdown of c-Abl rendered HeLa cells less sensitive to MX. The effect of imatinib was decreased by c-Abl siRNA suggesting a role for catalytically inactive c-Abl in the death cascade. These findings indicate that MX has a unique cytotoxic effect when the kinase activity of c-Abl is inhibited. The treatment results in increased DNA damage and c-Abl-dependent apoptosis, which may offer new possibilities for potentiation of cancer chemotherapy.
Proceedings of the National Academy of Sciences, 2000
In over 90% of cervical cancers and cancer-derived cell lines, the p53 tumor suppressor pathway i... more In over 90% of cervical cancers and cancer-derived cell lines, the p53 tumor suppressor pathway is disrupted by human papillomavirus (HPV). The HPV E6 protein promotes the degradation of p53 and thus inhibits the stabilization and activation of p53 that would normally occur in response to HPV E7 oncogene expression. Restoration of p53 function in these cells by blocking this pathway should promote a selective therapeutic affect. Here we show that treatment with the small molecule nuclear export inhibitor, leptomycin B, and actinomycin D leads to the accumulation of transcriptionally active p53 in the nucleus of HeLa, CaSki, and SiHa cells. Northern blot analyses showed that both actinomycin D and leptomycin B reduced the amount of HPV E6-E7 mRNA whereas combined treatment with the drugs showed almost complete disappearance of the viral mRNA. The combined treatment activated p53-dependant transcription, and increases in both p21 WAF1͞CIP1 and Hdm2 mRNA were seen. The combined treatment resulted in apoptotic death in the cells, as evidenced by nuclear fragmentation and PARP-cleavage indicative of caspase 3 activity. These effects were greatly reduced by expressing a dominant negative p53 protein. The present study shows that small molecules can reactivate p53 in cervical carcinoma cells, and this reactivation is associated with an extensive biological response, including the induction of the apoptotic death of the cells.
Rapid and effective detection of mutations in the p53 gene using nonradioactive single-strand conformation polymorphism (SSCP) technique applied on PhastSystem™
Journal of Virological Methods, 1995
The polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) method is a powe... more The polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) method is a powerful tool for the screening of genetic alterations, including single-base substitutions. In the present study, the conventional SSCP technique was modified on the semiautomated electrophoresis system (PhastSystem) for the detection of mutations in the p53 tumor suppressor gene. The SSCP running conditions were optimized for three PCR-amplified DNA fragments, spanning exons 5 through 9 of the p53 gene, using the PCR-products derived from the CaSki and HaCaT cells as the normal and mutant controls, respectively. The optimized SSCP protocols were tested on nine human vulvar and vaginal carcinoma-derived cell lines. The optimizing experiments indicated that the running temperature and gel density can affect significantly the electrophoretic mobility and resolution of single-stranded DNA molecules. Because the gel temperature is the most important parameter affecting the conformation and thus electrophoretic mobility of single strands, one of the most important advantages of the SSCP technique on the PhastSystem is that the running temperature is controlled precisely. In addition to the fast electrophoretic separation, the PhastSystem also offers the use of a silver staining method allowing direct visualization of DNA with high detection sensitivity. Thus, the important advantage of this modified SSCP technique is the short time required for analysis, including electrophoresis and DNA detection. It is concluded that the SSCP method applied on the PhastSystem has the advantages of simplicity, efficiency, speed and reproducibility, and is suitable for clinical diagnostic purposes.
International Journal of Gynecological Cancer, 2005
Exogenous sex hormones are widely used by women either for pregnancy prevention, as part of infer... more Exogenous sex hormones are widely used by women either for pregnancy prevention, as part of infertility treatment, or for treatment of menopausal symptoms. The role of these hormones in the development of ovarian cancer has been vastly explored. The protective effect of combined oral contraceptive pill is confirmed in multiple studies, but it is not clear whether this protection also covers women with a genetic predisposition to ovarian cancer. There is no conclusive evidence of infertility treatments increasing ovarian cancer risk, but infertility as such is a risk factor. Currently available data suggest that long-term users of hormone replacement therapy may have a slightly increased risk for ovarian cancer compared to women who have never used estrogen. The risk might particularly involve the endometrioid type of ovarian cancer. Most data on ovarian cancer and estrogen comes from epidemiological studies, since the normally high concentrations of estrogens in ovarian tissue and follicular fluid make direct biologic studies on the effects of exogenous estrogens on the ovarian cell difficult. This review discusses the risk of ovarian cancer associated with the use of sex steroid hormones, with special emphasis on the possible risk associated with estrogens.
Anti-proliferative effect of retinoids and interferon-α-2a on vaginal cell lines derived from squamous intra-epithelial lesions
International Journal of Cancer, 1998
A panel of retinoids (all-trans-, 13-cis-, 19-cis retinoic acid and acitretin), and interferon-al... more A panel of retinoids (all-trans-, 13-cis-, 19-cis retinoic acid and acitretin), and interferon-alpha-2a was tested for the capacity to modulate the proliferation of UT-DEC-1 (HPV-33-positive) and UT-DEC-2 (HPV-16-positive) cell lines derived from vaginal intra-epithelial neoplasias (VAIN). At concentrations 10(-6) to 10(-8) M, all retinoids inhibited the growth of early-passage UT-DEC cell lines, but also of normal vaginal keratinocytes and fibroblasts. The inhibition was significantly reduced in late-passage UT-DEC cells. The effect on proliferation was essentially equal for all retinoids in high (1.8 mM)-Ca2+ medium, but decreased markedly in low (0.09 mM)-Ca2+ medium. Interferon-alpha-2a at 1000 IU/ml had an additive growth-inhibitory effect in the low- and in the high-Ca2+ medium. No consistent decrease in HPV E6-E7 mRNA levels could be associated either with retinoid or with interferon effect in either cell line. The expression of TGFbeta1 and TGFbeta2 mRNA increased 2- to 3-fold by 10(-6) M 13-cis-RA treatment in early- and in late-passage cells of both cell lines. TGFbeta1 at 0.1 to 1.0 ng/ml also inhibited the proliferation of both cell lines, and was more effective at early passage, but the inhibition was not dependent on calcium concentration. Neutralizing anti-TGFbeta antibodies partially relieved the proliferation inhibition by 13-cis-RA. The results show that the calcium-associated regulation of growth by the tested retinoids was seen in normal vaginal cells and in early pre-neoplastic cells, but was significantly reduced in cells with higher-grade phenotype, while also suggesting that the loss of responsiveness to retinoids and TGFbeta may play a role in the progression of squamous intra-epithelial neoplasia.
p53Mutations and Presence of HPV DNA Do Not Correlate with Radiosensitivity of Gynecological Cancer Cell Lines
Gynecologic Oncology, 1998
The correlation between p53 tumor suppressor gene mutations and the presence of high-risk human p... more The correlation between p53 tumor suppressor gene mutations and the presence of high-risk human papillomavirus (HPV) DNA with the in vitro radiosensitivity of gynecological malignancies was studied in 26 cell lines derived from gynecological cancers of 23 patients. Comparison of the intrinsic radiosensitivity was performed with mean inactivation dose (D) determined with the 96-well plate clonogenic assay. p53 mutations were investigated with polymerase chain reaction and single-strand conformation polymorphism (PCR-SSCP) analysis and direct DNA sequencing, and the presence of HPV DNA was studied with PCR using HPV consensus primers. p53 mutations were found in 6 of 10 vulvar squamous cell carcinoma (SCC) lines. Nine vulvar and 1 vaginal SCC cell lines were HPV DNA negative and 1 vulvar cell line was HPV 16 positive. All 4 cervical SCC lines were HPV positive and possessed the wild-type p53. Three cell lines expressed HPV 16 and 1 HPV 68. Among 10 endometrial cancer cell lines, 2 cell lines with mutant p53 and 1 HPV 16 positive cell line were found. No correlation could be demonstrated between inactivation of the p53 gene and radiosensitivity in vitro; the cell lines were evaluated as one group or according to their anatomical origin or histology. Our results indicate that inactivation of the p53 gene through mutation or binding with HPV DNA does not increase the resistance of gynecological malignancies to ionizing radiation in vitro.
Mutations of TP53 do not correlate with the sensitivity to paclitaxel—a study using 27 gynaecological cancer cell lines
European Journal of Cancer, 2002
The correlation between inactivation of the TP53 gene through mutation or the presence of high-ri... more The correlation between inactivation of the TP53 gene through mutation or the presence of high-risk human papillomavirus (HPV) DNA and intrinsic paclitaxel sensitivity was studied in 27 gynaecological cancer cell lines. IC(50) values, as a measure of drug sensitivity, were determined using a 96-well clonogenic assay. TP53 mutations were investigated with polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) and direct DNA sequencing. HPV status was studied with PCR using HPV consensus primers. TP53 mutations were found in 7/11 vulvar SCC cell lines. Only 2/9 endometrial and 1/7 ovarian cancer cell lines carried TP53 mutations. One vulvar and one endometrial cancer cell line were HPV-positive; both carrying HPV type-16 DNA. Thus, TP53 was functionally normal in 3/11 vulvar, 6/9 endometrial and 6/7 ovarian cancer cell lines. The IC(50) values for paclitaxel were 0.60-2.9, 0.49-2.3 and 0.40-3.4 nM in the vulvar, endometrial and ovarian cancer cell lines, respectively. No correlation could be demonstrated between inactivation of the TP53 gene and paclitaxel sensitivity in vitro; the cell lines were evaluated as one group or according to their anatomical origin or histology. Previous reports have given inconclusive results, partly due to the cell types used, i.e. normal, cancerous or transformed cells. Our results support the view that paclitaxel sensitivity of tumour-derived cancer cell lines is not related to the TP53 status.
The American Journal of Pathology, 1999
Collagenase-3 (MMP-13) is a human matrix metalloproteinase specifically expressed by invading tum... more Collagenase-3 (MMP-13) is a human matrix metalloproteinase specifically expressed by invading tumor cells in squamous cell carcinomas (SCCs) of the head and neck. Here, we have further elucidated the role of MMP-13 in tumor invasion by examining its expression in invasive malignant tumors of the female genital tract. Using in situ hybridization, expression of MMP-13 mRNA was detected in 9 of 12 vulvar SCCs, primarily in tumor cells, but not in intact vulvar epithelium, in cervical SCCs (n = 12), or in endometrial (n = 11) or ovarian adenocarcinomas (n = 8). MMP-13 expression was especially abundant in vulvar carcinomas showing metastasis to lymph nodes and was associated with expression of membrane type 1 MMP by tumor cells and gelatinase-A (MMP-2) by stromal cells, as detected by immunohistochemistry. MMP-13 mRNAs were detected in 9 of 11 cell lines established from vulvar carcinomas and in 4 of 6 cell lines from cervical carcinomas, whereas endometrial (n = 10) and ovarian (n = 9) carcinoma cell lines were negative for MMP-13 mRNA. No correlation was detected between MMP-13 expression and p53 gene mutations in vulvar SCC cell lines. However, MMP-13 expression was detected in 5 of 6 vulvar and cervical SCC cell lines harboring HPV 16 or 68 DNA. These results show that MMP-13 is specifically expressed by malignantly transformed squamous epithelial cells, including vulvar SCC cells, and appears to serve as a marker for their invasive capacity.
Mutation of tumor suppressor gene p53 is frequently found in vulvar carcinoma cells
American Journal of Obstetrics and Gynecology, 1995
The purpose of this study was to evaluate the presence and type of mutations of the tumor suppres... more The purpose of this study was to evaluate the presence and type of mutations of the tumor suppressor gene p53 in squamous carcinoma cell lines of the vulva. Eight low-passage cell lines established from vulvar carcinoma were included in the analysis. Mutational analysis was restricted to exons 5 through 9 of the p53 gene, previously shown to have a high incidence of mutations. The sequences containing exons 5/6,7, and 8/9 were amplified by polymerase chain reaction and screened with a single-strand conformation polymorphism technique on PhastSystem (Pharmacia Biotech, Uppsala, Sweden). Exons from samples showing mobility shifts in single-strand conformation polymorphism were sequenced by polymerase chain reaction direct sequencing. Five vulvar carcinoma cell lines showed abnormal electrophoretic mobility of exons 5/6, one of exons 8/9, and one of exon 7. Reduction to homozygosity was detected in four vulvar carcinoma cell lines. Missense mutations were detected by sequence analysis in UM-SCV-2 (codon 171: GAG[Glu]--&gt;TAG[STOP]), UM-SCV-3 (hot spot codon 273: CGT[Arg]--&gt;TGT[Cys]), UM-SCV-4 (codon 151: CCC[Pro]--&gt;CAC[His]), UM-SCV-5 (codon 155: ACC[Thr]--&gt;ATC[lle]), and UM-SCV-7 (codon 245: GGC[Gly]--&gt;AGC[Ser]). UM-SCV-3 also carried a missense mutation with no amino acid change (codon 314: TCC[Ser]--&gt;TCT[Ser]). UM-SCV-7 carried an additional base deletion at codon 249 (AGG--&gt;AG-), likely resulting in a frameshift in transcription and a truncated protein product. Four of the seven mutations were transitions, two were transversions, and one was a deletion. The presence of transitions suggests that at least a proportion of p53 mutations of these cancers may arise spontaneously without exogenous carcinogen exposure. UM-SCV-1A and UM-SCV-1B were derived from the primary tumor and pleural effusion of the same patient. UM-SCV-6 is a cell line that contains human papillomavirus 16. No mutations in these three cell lines were found by single-strand conformation polymorphism. On the basis of previous observations, loss of tumor suppressor p53 function either by mutation or human papillomavirus involvement is a frequent phenomenon in cervical carcinoma cells. It appears now that functional inactivation of p53 is associated also with vulvar carcinoma cell lines, but mutations of the p53 gene are much more common in vulvar than in cervical carcinoma cell lines.
International Journal of Cancer, 1992
Explant cultures were started from human papillomavirus (HPV)-infected genital lesions in order t... more Explant cultures were started from human papillomavirus (HPV)-infected genital lesions in order to isolate and propagate abnormally differentiating cells from squamous intraepithelial neoplasia. A medium with high calcium concentration was used to induce terminal differentiation of cells from surrounding normal epithelium. Two cell lines with extended life-spans were established. The UT-DEC-1 cell line was derived from an HPV-33-positive mild vaginal dysplasia (VAIN I). In cultured UT-DEC-1 cells, HPV 33 DNA was detected with Southern-blot hybridization and the polymerase chain reaction (PCR) technique. The restriction pattern of HPV 33 changed during early passages and flow cytometric analysis detected a decrease in chromosomal DNA content. HPV 33 RNA from the E6-E7 region could be amplified by PCR at late passage. UT-DEC-2 cell line was derived from an HPV-16-positive moderate vaginal dysplasia (VAIN II). HPV 16 DNA was also detected in cultured cells by the PCR technique. The senescence of normal keratinocytes and growth selection in favor of aneuploid cells was observed by flow cytometric analysis at subsequent passages. Karyotype analysis showed clonal chromosomal abnormalities in both cell lines. To date, UT-DEC-1 cells have undergone 40 and UT-DEC-2 cells 25 passages. This study shows that the isolation of HPV-infected dysplastic cells can be achieved by culturing the cells in a medium with high calcium concentration. The cell lines presented provide the opportunity of evaluating the early stages of squamous-cell carcinogenesis.
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Papers by Sakari Hietanen