Correction to: Recurrent pregnancy loss is associated to leaky gut: a novel pathogenic model of endometrium inflammation?

Following publication of the original article [1], the authors reported updated affiliations for five of the authors. The updated affiliations are shown below and reflected in the affiliation list of this Correction.

Recurrent pregnancy loss is associated to leaky gut: a novel pathogenic model of endometrium inflammation?

BACKGROUND: Recurrent pregnancy loss (RPL) occurs in 3-5% in about 30% of cases no cause can be found. Women with RPL show higher prevalence of undiagnosed gut disorders. Furthermore, in endometrial tissues of RPL women, higher expression of pro-inflammatory cytokines and Nalp-3 inflammasome has been observed. Aim of this study was to investigate whether an abnormal gut permeability might occur in RPL women and allow passage into systemic circulation of pro-inflammatory molecules able to induce endometrial inflammation. METHODS: 70 women with idiopathic RPL and 30 healthy women were recruited at the Recurrent Pregnancy Loss Outpatient Unit of the Gemelli Hospital of Rome from March 2013 to February 2017. Enrolled women underwent 51Cr-ethylene-diamine-tetraacetic acid absorption test to evaluate intestinal permeability. Sera obtained from enrolled women were analysed for lipopolysaccharide (LPS) by ELISA. Anxiety and depression state were evaluated by administering STAI-Y and Zung-SDS tests, respectively. Of all recruited individuals, 35 women with idiopathic RPL and 20 healthy controls accepted to undergo diagnostic hysteroscopy and endometrial biopsy. Endometrial lysates were investigated for inflammasome Nalp-3 by Western blot analysis, and caspase-1, IL-1ß and IL-18 by ELISA, respectively. RESULTS: Higher prevalence of abnormal intestinal permeability (P < 0.0001), increased circulating levels of LPS (P < 0.05), anxiety (P < 0.05) and depression (P < 0.05) were observed in RLP women compared to controls. Endometrial expression of Nalp-3, caspase-1 and IL-1ß was significantly increased in RPL group (P < 0.0001; P < 0.05 and P < 0.001, respectively). IL-18 endometrial levels were not found to be higher in RPL cases. Statistically significant association between higher intestinal permeability and abnormally increased expression of endometrial Nalp-3, was observed in RPL (P < 0.01). Furthermore, higher LPS serum levels, a bacterial-derived activator of Nalp-3 complex, was shown to be statistically associated to abnormal endometrial expression of Nalp-3 inflammasome (P < 0.01) in RPL women. CONCLUSIONS: In women with RLP, leaky gut might occur and allow passage into circulation of immune triggers, potentially able to elicit endometrial innate immune response and, thus, to contribute to miscarriage pathogenesis. Diagnosis and treatment of intestinal disorders underlying leaky gut might improve endometrial environment and pregnancy outcome.

Phase II study of NGR-hTNF in combination with doxorubicin in relapsed ovarian cancer patients.

BACKGROUND: The NGR-hTNF (asparagine-glycine-arginine-human tumour necrosis factor) is able to promote antitumour immune responses and to improve the intratumoural doxorubicin uptake by selectively damaging tumour blood vessels. METHODS: Patients progressing after ≥ 1 platinum/taxane-based regimen received NGR-hTNF 0.8 µg m(-2) and doxorubicin 60 mg m(-2) every 3 weeks. Primary endpoint was a Response Evaluation Criteria in Solid Tumors-defined response rate with a target of more than 6 out of 37 responding patients. RESULTS: A total of 37 patients with platinum-free interval lower than 6 months (PFI<6; n=25), or between 6 and 12 months (PFI=6-12; n=12) were enrolled. Median baseline peripheral blood lymphocyte count (PBLC) was 1.6 per ml (interquartile range, 1.2-2.1). In all, 18 patients (49%) received more than 6 cycles. Febrile neutropaenia was registered in one patient (3%). Among 35 assessable patients, 8 (23%; 95% CI 12-39%) had partial response (2 with PFI<6; 6 with PFI=6-12) and 15 (43%) had stable disease (10 with PFI<6; 5 with PFI=6-12). Median progression-free survival (PFS) was 5.0 months for all patients, 3.8 months for patients with PFI<6, and 7.8 months for patients with PFI=6-12. Median overall survival (OS) was 17.0 months. Patients with baseline PBLC higher than the first quartile had improved PFS (P=0.01) and OS (P=0.001). CONCLUSION: Tolerability and activity of this combination warrant further randomised testing in patients with PFI<6. The role of PBLC as a blood-based biomarker deserves further investigation.

Phase II study on weekly bolus topotecan in advanced or recurrent cervical cancer.

OBJECTIVE: It was the aim of our study to evaluate the efficacy and safety of weekly topotecan in patients with advanced or recurrent cervical disease. METHODS: Topotecan was administered intravenously as a weekly infusion at a dose of 3.5 mg/m(2) on days 1, 8 and 15 of a 28-day cycle. After the second cycle, the dose was increased to 4 mg/m(2) if no grade >2 toxicity occurred. Treatment was continued until disease progression or unacceptable toxicity. RESULTS: Twenty-one patients were enrolled, but only 18 were evaluable for response and toxicity. Ten patients (56%) had received primary surgery + chemoradiation, 6 patients (33%) had previously received surgery + chemotherapy and 2 patients (11%) exclusive chemoradiation. Patients received a mean of 3.5 courses (range 1-6). No complete or partial responses were reported. Two patients (11%) presented disease stabilization as maximum response. Median progression-free survival was 11 weeks (95% CI 15-25), and median overall survival was 28 weeks (95% CI 24-72). The treatment was generally well tolerated. CONCLUSIONS: This trial did not report any activity of weekly bolus topotecan in the treatment of advanced or recurrent cervical cancer. Actually, there is no evidence to recommend this therapy in this patient population.

UbcH10 expression may be a useful tool in the prognosis of ovarian carcinomas.

The UbcH10 gene codes for a protein that belongs to the ubiquitin-conjugating enzyme family. Previous studies of our group suggest UbcH10 expression as a valid indicator of the proliferative and aggressive status of thyroid carcinomas. Therefore, to better understand the process of ovarian carcinogenesis, and to look for possible tools to be used as prognostic markers in these neoplasias, we decided to extend the analysis of the UbcH10 expression to the ovarian neoplastic disease. We found that the UbcH10 gene was upregulated in some ovarian carcinoma cell lines analysed. Then, immunohistochemical studies demonstrate that UbcH10 expression significantly correlates with the tumor grade and the undifferentiated histotype of the ovarian carcinomas. Furthermore, a significant relationship between UbcH10 expression and overall survival was observed. Finally, the block of UbcH10 protein synthesis by RNA interference inhibited the growth of ovarian carcinoma cell lines, suggesting a role of UbcH10 overexpression in ovarian carcinogenesis. Therefore, all these data taken together suggest the possibility to use UbcH10 detection as a marker for the diagnosis and prognosis of these neoplastic diseases and open the perspective of a therapy of some ovarian carcinomas based on the suppression of the UbcH10 synthesis and/or function.

RB family members as predictive and prognostic factors in human cancer.

The retinoblastoma family members--pRb, pRb2/p130 and p107--are tumor suppressor genes involved in controlling four major cellular processes: growth arrest, apoptosis, differentiation and angiogenesis. Molecular genetic studies have identified abnormalities of these tumor suppressor genes in a large proportion of human cancers. These genetic alterations have emerged as significant factors in the pathogenesis and progression of many types of tumors and are therefore likely to provide relevant information to assess risk in cancer patients. There is a pressing clinical need to identify prognostic and predictive factors for patients with cancer, because there is an undeniable importance in being able to determine which patients will have a favorable outcome without further therapy (prognostic factor) and which will need some additional treatment (predictive factor). This review examines the predictive and/or prognostic role of each retinoblastoma family member in human cancer.

Frequent loss of expression of the cyclin-dependent kinase inhibitor p27 in epithelial ovarian cancer.

p27Kip1 is a member of the Cip1/Kip1 family of cyclin-dependent kinase inhibitors and is a potential tumor suppressor gene. Low levels of p27 are associated with poor prognosis in a variety of tumors, including breast, colon, prostate, and lung carcinomas. In the present study, p27 protein expression was investigated by immunohistochemistry and Western blot analysis in a series of 82 epithelial ovarian tumors [16 classified as low malignant potential (LMP) and 66 classified as primary ovarian adenocarcinomas]. Immunohistochemical analysis revealed frequent loss of p27 expression in primary ovarian adenocarcinomas (33%), with respect to LMP tumors (6%; P = 0.0009). In addition to nuclear staining, cytoplasmic localization of p27 was noted in 45 (55%) of 82 cases. p27 levels inversely correlated with cdk2 kinase activity in a representative subset of tumors. When the clinical outcome of the patients was evaluated in relationship to p27 status, we observed a significant correlation between presence of p27 staining and a longer time to progression (P = 0.032 by log-rank test). These data indicate that loss of p27 is a frequent event in ovarian carcinomas as compared with LMP tumors, suggesting that these tumor types may have different pathogenesis. p27 levels may also represent a useful prognostic marker for predicting disease recurrence in primary ovarian carcinomas.

Cell cycle genes as targets of retinoid induced ovarian tumor cell growth suppression.

We have examined the effect of all-trans-retinoic acid (RA) on cell cycle gene expression in RA sensitive CA-OV3 and RA resistant SK-OV3 ovarian carcinoma cell lines. Gene expression was analysed by multiprobe RNAse protection, Western blotting and in vitro kinase assays. No differences were observed between RA sensitive and RA resistant ovarian carcinoma cells in the levels of expression of many cell cycle genes including cyclin A, B and E, cdk 2,4 and 6, E2F-1, E2F-2, E2F-3, E2F-4, E2F-5, DP-1 and DP-2. However, RA sensitive CA-OV3 cells expressed higher levels of p53, p27, p21, and p16 compared to RA resistant SK-OV3 cells. In addition, RA treatment of CA-OV3 cells resulted in a significant decrease in hyperphosphorylated RB and RB-2/p130 and corresponding significant increases in the levels of hypophosphorylated and/or partially phosphorylated RB-2/p130 protein and hypophosphorylated RB. Also, RA treatment increased expression of the cdk inhibitor p27 and decreased activity of cdk 2, cdk 4 and cdk 6. Finally, amounts of p27-cyclin E and RB-2/p130-E2F4 complexes were found to increase in CA-OV3 cells growth arrested by RA. These results suggest that the pocket protein pathways are critical targets for retinoid suppression of ovarian carcinoma cell growth.

p27Kip1 expression is associated with clinical outcome in advanced epithelial ovarian cancer: multivariate analysis.

Few biological parameters have been shown to have a prognostic role in patients with advanced ovarian cancer. p27Kip1 is a cyclin-dependent kinase inhibitor, and its loss may contribute to tumor progression. We determined whether p27Kip1 protein expression in advanced ovarian cancer could be associated with prognosis. p27Kip1 status was assessed by immunohistochemical analysis of tissue sections from primary tumors of 99 patients with stages III-IV ovarian carcinoma and was analyzed in relation to clinicopathological variables, time to progression (TTP), and overall survival (OS). p27Kip1 expression was detected in 47 (47%) of the 99 patients. p27 expression did not correlate with any of the classical clinicopathological parameters. Loss of p27 protein was significantly associated with a short TTP (P = 0.0004) and decreased OS (P = 0.0302). The 5-year TTP rate in p27-positive patients was 50% versus 11% in p27-negative patients. p27-positive cases showed a 5-year OS rate of 53% compared with 43% of p27-negative cases. In multivariate analysis, p27 expression was an independent predictor of progression of disease (P = 0.0009) and survival (P = 0.0032) when considered together with stage of disease, presence of ascites, and residual tumor at surgery. Loss of p27Kip1 conferred poor prognosis independently of proliferative index, as assessed by proliferating cell nuclear antigen immunostaining. p27 immunoreactivity can be used to predict progression of disease and survival in patients with advanced epithelial ovarian cancer and therefore may represent a new prognostic marker.

The Rb family of cell cycle regulatory factors: clinical implications.

The retinoblastoma gene family is composed of three members: the product of the retinoblastoma gene (pRb), which is one of the most well studied tumor suppressor genes and two related proteins, pRb2/p130 and p107, which have been shown to be structurally and functionally similar to pRb. The three retinoblastoma family members show growth suppressive properties, although the growth arrest mediated by each of the three pocket regions of the proteins is not identical. This supports the idea that although the three members may complement each other, they are not fully functional or redundant. Among the three family members, the retinoblastoma-related gene product pRb2/p130 is a tumor suppressor gene and an effective candidate target for gene therapy approach. The aim of this review is to examine the role of the Rb family members in growth regulation discussing their putative prognostic and therapeutical impact in human cancer.

Expression of the retinoblastoma-related gene Rb2/p130 is downregulated in atypical endometrial hyperplasia and adenocarcinoma.

The retinoblastoma-related gene Rb2/p130 encodes a protein that is a negative cell-cycle regulator normally expressed in a number of adult tissues. This protein shares many structural and functional features with the product of the retinoblastoma gene, one of the best-studied tumor-suppressor genes, and plays a fundamental role in growth control. The Rb2/p130 gene product associates with specific members of the E2F family and various cyclins, displaying a growth-suppressive activity specific for the G(0)/G(1) phases. It has been reported that Rb2/p130 is involved in the pathogenesis and progression of lung cancer and mesothelioma. We previously demonstrated for the first time that reduced immunohistochemical expression of Rb2/p130 was a strong independent predictor of poor outcome in endometrial cancer. The aim of the present study was to evaluate Rb2/p130 expression in normal, hyperplastic, and neoplastic endometrial lesions to determine whether the protein plays a significant role in endometrial carcinogenesis. We evaluated Rb2/p130 expression by immunohistochemistry staining in 102 specimens chosen to represent a spectrum of endometrial changes, including proliferative endometrium (n = 18), secretory endometrium (n = 18), simple or complex hyperplasia without atypia (n = 18), atypical hyperplasia (n = 18), and invasive carcinoma (n = 30). We found that Rb2/p130 was highly expressed in proliferative endometrium and in hyperplasia without atypia, the mean percentage of stained nuclei being 66% and 60%, respectively, but was downregulated in secretory endometrium, atypical hyperplasia, and carcinoma, with mean scores of 38%, 25%, and 22%, respectively. When categorized on a semiquantitative scale (negative v 1% to 50% v >50% positivity), endometrial cancer displayed significantly less staining than all other endometrial samples (P <.001). Poorly differentiated carcinomas (n = 9) showed a significantly lower immunoreactivity for Rb2/p130 than did well-differentiated carcinomas (n = 11; P =.005) and moderately differentiated carcinomas (n = 10; P =.03). In addition, atypical hyperplasia showed a significantly lower immunoreactivity than either proliferative endometrium (P =.003) or hyperplasia without atypia (P = 0.02). Our findings of a progressive decrease in Rb2/p130 expression from hyperplastic endometrium through atypical hyperplasia to poorly differentiated carcinomas suggest the involvement of this negative cell-cycle regulator in endometrial carcinogenesis. Furthermore, immunostaining for Rb2/p130 may prove diagnostically useful in the often difficult distinction between hyperplastic and atypical hyperplastic endometrium. HUM PATHOL 32:360-367.

Expression of cell-cycle-associated proteins pRB2/p130 and p27kip in vulvar squamous cell carcinomas.

Squamous cell vulvar carcinoma accounts for 4% of all gynecologic malignancies. The cause of vulvar cancer is still unclear. Identification of new biologic factors involved in vulvar carcinogenesis may be useful in clarifying the natural history of this malignancy. We investigated the immunohistochemical expression of the retinoblastoma-related proteins pRB2/p130 and CKI p27kip1 in a series of 51 invasive squamous cell carcinomas of the vulva (ISCCs) and in synchronous normal vulvar skin, non-neoplastic epithelial disorders (NNED) and vulvar intraepithelial neoplasia (VIN). Normal vulvar skin staining showed positivity for both pRB2/p130 and p27kip1. Loss of pRB2/p130 occurred in 29 (57%) of 51 specimens of ISCCs, and in 1 of 7 specimens with VIN (14%; P = .04). We also observed a significant decrease of pRB2/p130 expression from NNED to neoplastic tissues (VIN and ISCCs) (P = .004). Loss of p27kip1 expression was found in 16 of 51 specimens (31%) of invasive carcinomas, in 1 (14%) of 7 specimens of VIN, and in 2 of 18 specimens of NNED (11%). pRB2/p130 and p27(kip1) did not correlate significantly with any of the clinicopathologic parameters examined. Our data indicate that loss of pRB2/p130 and p27kip1 are frequent events in invasive vulvar carcinomas compared with synchronous premalignant lesions, non-neoplastic epithelial disorders, and normal vulvar skin. The significant progressive decrease of pRB2/p130 expression from non-neoplastic epithelial alterations through intraepithelial neoplasia to invasive vulvar carcinomas suggests a role for this tumor suppressor gene in vulvar carcinogenesis.

[A zinc sulfate and usnic acid preparation used as post-surgical adjuvant therapy in genital lesions by Human Papillomavirus]. / Terapia adiuvante con un preparato a base di zinco solfato e acido usnico delle lesioni genitali da Human Papilloma virus (HPV) dopo trattamento chirurgico distruttivo.

BACKGROUND: To evaluate the efficacy and safety of intravaginal administration of a zinc sulphate and usnic acid compound as adjuvant therapy of Human Papillomavirus (HPV) genital infection, after radiosurgical treatment (RS). METHODS: One hundred patients affected by HPV genital infection were enrolled in the study from October 1996 to July 1998. Patients were classified according to colposcopic and cytologic criteria and treated with RS. Patients were randomized into three groups: the first group did not follow any therapy after RS (control group), (n = 50); the second group was pharmacologically treated with intravaginal administration of a usnic acid and zinc sulphate compound (Zeta N, Bergamon Italia) before and after RS (n = 25), the third group was pharmacologically treated only after RS (n = 25). The last two groups were considered together for the statistical analysis. Patients were reevaluated after one, two, three and six months from electrocoagulation. The safety of treatment was also investigated. RESULTS: One month after RS. HPV lesions disappeared in 93% of the patients in the control group and in 100% of patients treated with usnic acid and zinc sulphate. After one month, reepithelization was complete in 65% of cases treated with usnic acid and zinc sulphate and in only 28% of the control group (p = 0.001). Two months later reepithelization was 94% in the patients pharmacologically treated compared to 76% of the control group (p = 0.06). Treatment prior to RS resulted in a reduction of the overall area of lesions in 88% of cases. Three months after RS, there was a significant reduction of recurrence in the group treated with usnic acid and zinc sulphate (p = 0.01). This reduction was still significant at six months (p = 0.005). CONCLUSIONS: Usnic acid and zinc sulphate adjuvant treatment improved time of reepithelization and reduce the recurrence with few side effects and a good compliance.

[Therapy of bacterial vaginosis. Prospective clinical study ot the efficacy and tolerability of quaternary ammonium salts vs. clindamycin]. / La terapia delle vaginosi batteriche. Studio clinico prospettico sull'efficacia e tollerabilità dei sali quaternari d'ammonio versus clindamicina.

BACKGROUND: To evaluate the efficacy and safety of intravaginal quaternary ammonium antimicrobial compounds (SQA) versus clindamycin 2% intravaginal cream (CL) in the treatment of bacterial vaginosis (VB). MATERIALS AND METHODS: One hundred-thirty-three patients affected by VB were enrolled in the study from January 1995 to October 1997. Patients were classified according to Amsel's criteria and/or to the indications of the Scandinavian Society of Bacterial Vaginosis. Twenty-three patients were initially excluded from the study, and 110 patients were randomized in two groups, SQA versus CL. Patients were reevaluated after 3 weeks, 3 months and 6 months from the end of therapy. The safety of treatment was also investigated. RESULTS: Of 110 patients, 59 were treated with SQA and 51 with CL. One hundred (90.9%) patients completed the therapy and were subjected to the first control after 3 weeks from the end of therapy. A significant reduction of most of the symptoms and all signs of VB was observed in the group treated with SQA. Similarly, a significant reduction of most of the symptoms (vaginal and urinary in particular) and all signs of VB was observed in the group treated with CL. The percentage of response was 86.7% for SQA group and 87.2% for CL group. Moreover, after 3 months from the end of therapy, 47.2% and 50% of the patients treated with SQA and CL, respectively, recurred, and after 6 months 78.5% and 75% of the patients recurred, respectively. CONCLUSIONS: SQA treatment conferred 86.7% of response after 3 weeks from the end of therapy, with poor side effects and a good compliance in good keeping with the results obtained with CL treatment.

Lethal Clostridium difficile Colitis Associated with Paclitaxel and Carboplatin Chemotherapy in Ovarian Carcinoma: Case Report and Review of the Literature.

Clostridium difficile colitis, although rare, could represent a serious complication following chemotherapy. Prior antibiotic use has been considered the single most important risk factor in the development of C. difficile infection. Recently, the association between antineoplastic therapy and C. difficile-associated diarrhea in the absence of a prior antibiotic therapy has become more apparent. A 75-year-old woman with serous adenocarcinoma of the ovary developed lethal pancolitis caused by C. difficile after five cycles of paclitaxel- and carboplatin-based chemotherapy. She presented with diarrhea, coffee-ground emesis, and oliguria and was hospitalized immediately for aggressive treatment. Despite all the medical efforts, her condition worsened and she died after twenty days. We describe the second case reported of a patient developing a severe C. difficile colitis following chemotherapy without any recent antibiotic use and review the data of the literature, emphasizing the need to a prompt diagnosis and management that can significantly decrease the morbidity and life-threatening complications associated with this infection.

Controversies in the management of endometrial cancer.

Endometrial cancer (EC) remains the most common malignancy of the female genital tract. The median age at diagnosis is the sixth decade, with abnormal uterine bleeding at the presentation in 90% of the patients. Surgical treatment, including complete hysterectomy, removal of remaining adnexal structures, and an appropriate surgical staging, represents the milestone of curative therapy for patients with EC. Adjuvant therapy is necessary in patients at high risk of recurrence. Conservative treatment approaches should be used in selected cases for women with a desire of fertility preservation. This review summarizes the management of EC and discusses current controversies regarding the role of lymphadenectomy and radiotherapy in patients with intermediate-risk tumors confined to the uterus.

Importance of a specialized pathologist for the examination of frozen sections of adnexal masses.

The aim of the present study was to examine the impact of a specialized pathologist on the accuracy of frozen section analysis for adnexal masses. We included women who underwent frozen section diagnosis for adnexal mass surgery. A specialized and a general pathologist read the sections. We calculated sensitivity, specificity, positive and negative predictive values, and accuracy for the whole series, as well as for the specialized and general pathologist groups. We included 325 patients; in 103 patients (31.7%), frozen section diagnosis was performed by the specialized and in 222 (68.3%), by the general pathologist. There was a significant difference in terms of correspondence between the specialized and the general pathologist groups (P= 0.024). We registered four overdiagnoses (both performed by the general pathologist [1.8% vs 0%]) and 56 underdiagnoses of which 14 (13.6%) were made by the specialized pathologist and 46 (20.7%) by the general pathologist. In 14 cases (4.3%), diagnosis could not be made on frozen section and was postponed to final histology for definitive diagnosis (1/103 [0.9%] for the specialized pathologist and 13/222 [5.8%] for the general pathologist). Our data confirm previous reports on the accuracy of frozen section analysis of adnexal masses and show a significant positive impact of the specialized pathologist as opposed to the general pathologist.

Endometrioid adenocarcinoma with squamous differentiation arising from ureteral endometriosis in a patient with no history of gonadal endometriosis.

BACKGROUND: Malignant transformation may occur in up to 1% of women with endometriosis and only 20% of these cases occur at extragonadal pelvic sites. Similarly, urinary tract endometriosis is rare and occurs in only 1% of all endometriotic lesions. CASE: An endometrioid carcinoma with squamous differentiation arising from periureteral endometriosis presented as a pelvic mass encasing the right ureter. The patient had a history of total hysterectomy and bilateral salpingo-oopherectomy 8 years earlier because of bilateral serous benign cysts with right hemorrhagic corpus luteum and uterine fibroids, and has since been under unopposed estrogen replacement therapy for 5 years. CONCLUSION: This is the second case of a malignancy arising in endometriosis presenting as an obstructive ureteral mass and the first case of a patient with this condition whose endometriosis is not consistent with a gonadal origin. An analysis of the case and related literature is presented together with the discussion of possible pathogenetic mechanisms.

Paclitaxel induces apoptosis in Saos-2 cells with CD95L upregulation and Bcl-2 phosphorylation.

We examined the effect of paclitaxel on human osteoblastic cells Saos-2 to determine if paclitaxel can affect proliferation and apoptosis. We used a p53-negative cell line in order to mimic the loss of function frequently observed at the clinical level. Paclitaxel induced cell death in a dose- and time-dependent manner. Marked nuclear condensation and fragmentation of chromatin were observed by Hoechst 33258 stain, DNA ladder formation, electron microscopy, and flow cytometry at concentrations as low as 100 nM, a concentration which can be achieved by infusion in human plasma. At 100 nM, paclitaxel induced a G2 arrest at 8 h of treatment. The cells then continued to accumulate in G2 until 72 h when the percentage of apoptotic events reached 54%. At the molecular level, Bcl-2 protein was phosphorylated at 16 h and PARP protein was cleaved, indicating the activation of caspase-3-like proteases. Caspase inhibitors Z-VAD-FMK and Z-DEVD-FMK rescued Saos-2 cells from paclitaxel-induced apoptosis. CD95 expression was constantly high, while CD95L showed a threefold increase in expression. This suggests that, following the G2 arrest, apoptosis is induced through the CD95/CD95L system.