Sakari Hietanen

Activation of p53 in cervical cancer cells by human papillomavirus E6 RNA interference is transient, but can be sustained by inhibiting endogenous nuclear export-dependent p53 antagonists.

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.

Rapid and effective detection of mutations in the p53 gene using nonradioactive single-strand conformation polymorphism (SSCP) technique applied on PhastSystem™

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.

Mutation of tumor suppressor gene p53 is frequently found in vulvar carcinoma cells

OBJECTIVE 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. STUDY DESIGN 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. RESULTS 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]-->TAG[STOP]), UM-SCV-3 (hot spot codon 273: CGT[Arg]-->TGT[Cys]), UM-SCV-4 (codon 151: CCC[Pro]-->CAC[His]), UM-SCV-5 (codon 155: ACC[Thr]-->ATC[lle]), and UM-SCV-7 (codon 245: GGC[Gly]-->AGC[Ser]). UM-SCV-3 also carried a missense mutation with no amino acid change (codon 314: TCC[Ser]-->TCT[Ser]). UM-SCV-7 carried an additional base deletion at codon 249 (AGG-->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. CONCLUSIONS 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.

The cytotoxicity of chemotherapy drugs varies in cervical cancer cells depending on the p53 status

Metastatic cervical cancer remains a clinical problem. The development of more efficient treatment modalities and the optimal use of chemo- and radiotherapy require better understanding of their impact on regulation of cell survival and apoptosis, but the issue is insufficiently explored. Human papillomavirus (HPV) E6 protein is present in nearly all cervical cancers, targeting the p53 tumor suppressor protein for degradation. We studied the role of p53 in mediating the cytotoxic effects of 31 chemotherapy compounds. SiHa cervical cancer cells, carrying wild type (wt) p53 and HPV16 genome, were stably transfected with dominant negative p53 (DDp53) or ectopic HPV16 E6 in order to inhibit p53 function. The effects of chemotherapy drugs in these cells were compared to the effects in cells retaining endogenous residual p53 activity. 28 out of 31 drugs reduced the amount of E6 mRNA, but only some induced marked p53 activation. In clonogenic cell survival experiments, the presence of DDp53 and ectopic E6 either increased or decreased cytotoxicity, depending on the drug. The decrease of E6 mRNA was necessary, but not sufficient event in the p53 activation by chemotherapy. The impact of p53 on clonogenic cell survival varied between 0- 60%, indicating that p53 plays an important, but not crucial role in response to chemotherapy. The finding that chemosensitivity varies depending on the p53 status may have clinical implications, since early stage cervical cancer cells usually carry wt p53, whereas p53 mutations are frequently found in advanced disease.

Mutations of TP53 do not correlate with the sensitivity to paclitaxel--a study using 27 gynaecological cancer cell lines.

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.

Anti‐proliferative effect of retinoids and interferon‐α‐2a on vaginal cell lines derived from squamous intra‐epithelial lesions

A panel of retinoids (all‐trans‐, 13‐cis‐, 19‐cis retinoic acid and acitretin), and interferon‐α‐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‐α‐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 TGFβ1 and TGFβ2 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. TGFβ1 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‐TGFβ 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 TGFβ may play a role in the progression of squamous intra‐epithelial neoplasia. Int. J. Cancer 78:338–345, 1998.© 1998 Wiley‐Liss, Inc.

Response of estrogen receptor-positive intraabdominal fibromatosis to aromatase inhibitor therapy

BACKGROUND Intraabdominal fibromatosis is a rare tumor-like lesion of uncertain etiology. CASE A 49-year-old woman underwent abdominal hysterectomy and bilateral salpingooophorectomy in 1997 to treat uterine leiomyomata and ovarian fibromatosis. Postoperatively, she received estradiol 2 mg daily as hormone replacement therapy (HRT). In 2000, laparotomy performed for a large pelvic tumor revealed inoperable intra-abdominal fibromatosis. The tumor, which was positive for estrogen and progesterone receptors, resolved during aromatase inhibitor therapy. The first follow-up computed tomographic (CT) scan revealed that the tumor masses were significantly reduced in size, and successive CT scans revealed stable disease. CONCLUSION Intraabdominal fibromatosis that expresses estrogen and progesterone receptors may respond favorably to treatment with aromatase inhibitors.