Intra-operative fluorescence angiography is reproducible and reduces the rate of anastomotic leak after colorectal resection for cancer: a prospective case-matched study.

AIM: Intra-operative fluorescence angiography (IOFA) with indocyanine green provides information on tissue perfusion that may help prevent an anastomotic leak (AL). The aim of this study was to assess the impact of IOFA on outcomes after left-sided colonic or low anterior resection with anastomosis for colorectal cancer. METHODS: All patients with left-sided colonic or rectal cancer, operated between June 2017 and December 2018, were prospectively included. IOFA has been routinely implemented since May 2018. Reproducibility of IOFA, after a 1:1 matching for relevant clinical risk factors of AL, was studied in patients with IOFA (IOFA+) and without IOFA (IOFA-). Outcomes were compared in terms of postoperative events such as clinically relevant AL as the primary end-point. RESULTS: In the IOFA+ group, changing of the initially planned colon transection due to inadequate perfusion occurred in five out of 46 patients (10.9%). Agreement between intra-operative assessment and postoperative blind review of IOFA was deemed strong (Cohen's kappa index 0.893, 95% CI 0.788-0.998, P < 0.001). Among 111 patients, 42 matched patients were included in each group. There was significantly more clinically relevant AL in the IOFA- group compared to the IOFA+ group (16.7% vs 2.4%, P = 0.026) involving significantly more anastomotic dehiscence which required re-intervention (19% vs 2.4%, P = 0.014). Additionally, more descending colon ischaemia/necrosis was observed in the IOFA- group compared with the IOFA+ group (9.5% vs 0%, P = 0.040). CONCLUSION: In this prospective case-matched study, IOFA decreased the occurrence of clinically relevant AL due to necrosis of the descending colon or anastomosis. Upon blind review, perfusion assessment using IOFA was reproducible.

Acute appendicitis: Factors associated with inconclusive ultrasound study and the need for additional computed tomography.

PURPOSE: To identify variables associated with inconclusive ultrasound examination and the need for further abdominopelvic computed tomography (CT) examination for the diagnosis of acute appendicitis. MATERIALS AND METHODS: A total of 105 adult patients with acute appendicitis were included. There were 55 patients (38 men, 17 women; mean age, 23±9 [SD] years; range: 15-58 years) with a diagnosis of acute appendicitis using ultrasound alone and 50 patients (30 men, 20 women; mean age, 31±14 [SD] years; range: 16-83 years) who required further CT. Demographic, clinical, and biological criteria and appendix location were compared between the two groups to search for variables associated with the need of further CT. RESULTS: Patients who required further CT were older (31.1±14 [SD] years) and had a greater body mass index (BMI) (26.7±4.3 [SD]kg/m2) than those who did not require CT (23±9 [SD] years and 22.9±3.4 [SD]kg/m2), respectively (P<0.01). A greater proportion of patients with complicated acute appendicitis was observed in patients who required further CT (9/50; 18%) than in those who had only ultrasound (1/55; 2%) (P=0.012). Atypical appendix location was more frequent in patients who required CT (19/50; 36%) than in those who had only ultrasound (6/55; 11%) (P<0.001). There were no significant differences regarding gender, inflammatory syndrome and hours of imaging (on call vs. working hours) between the two groups. CONCLUSION: Advanced age, high BMI, atypical appendix location, and complicated appendicitis are associated with inconclusive ultrasound and the need for further CT to diagnose acute appendicitis.

Emergency surgery for obstructing colonic cancer: a comparison between right-sided and left-sided lesions.

PURPOSE: Few studies compare management and outcomes of obstructive colonic cancer, depending on the tumor site. We aim to evaluate the differences in patient characteristics, tumor characteristics, and outcomes of emergency surgery for obstructive right-sided versus left-sided colonic cancers. METHODS: Between 2000 and 2009, 71 consecutive patients had an emergency colectomy following strict and clear definition of obstruction in a single institution. We retrospectively analyzed pre, per, and postoperative data that were prospectively collected. RESULTS: There were 31 and 40 patients in the right and left group, respectively. Patients aged over 80 were more frequent in the right group (p = 0.03). At operation, ileocecal valve was less often competent in the right group (p = 0.03). The one-stage strategy was more frequent in the right group (p = 0.008). Patients in the right group had a higher rate of nodes invasion (p = 0.04). One- and two-year mortality rate in the right group had a tendency to be higher. CONCLUSIONS: Patients presenting with a right obstructive colonic cancer are older, have a more advanced locoregional disease, and are more often treated in a one-stage strategy than patients with a left obstructive tumor.

Combined Treatment of a Gallbladder Volvulus with a Common Bile Duct Obstruction.

Gallbladder volvulus is a rare disease and can lead to an acute cholecystitis. We report the case of an elderly woman with a gallbladder volvulus, diagnosed at CT scan and treated by surgery and endoscopic sphincterotomy.

Mesenteric panniculitis: still an ambiguous condition.

PURPOSE: To study the possible relationship between mesenteric panniculitis (MP) visible on computed tomography (CT) and the presence of an underlying neoplastic disease. PATIENTS AND METHODS: A retrospective analysis of 158 patients with CT examinations that revealed the presence of MP was performed. CT images were analyzed by two different radiologists using morphological criteria validated in the radiological literature. The presence, frequency and type of neoplastic lesions associated with MP were assessed. RESULTS: MP was asymptomatic in 96/158 patients (61%). Fat halo sign and pseudocapsule were visible on CT in 89/158 (56%) and 93/158 (59%) patients, respectively. Underlying neoplastic disease was present in 88/158 patients (56%). The neoplastic diseases most often associated with MP were lymphoma (28%), melanoma (18%), colorectal cancer (15%) and prostate cancer (13%). CONCLUSION: MP has typical CT appearance and is associated with underlying neoplastic disease in 56% of patients. Such levels of association might suggest that MP may be considered as a paraneoplastic condition. Hence, incidental depiction of MP on CT in a patient without known neoplastic disease should incite radiologists to further scrutinize CT examination for presence of synchronous neoplastic lesions.

Inflammatory appendix mass in patients with acute appendicitis: CT diagnosis and clinical relevance.

The purpose of our study was to analyze the clinical relevance of computerized tomography (CT) in providing the diagnosis of inflammatory appendix mass (IAM) in patients with acute appendicitis. The CT images of 134 patients were reviewed. Two groups of patients were made according to the presence (group 1; n = 21) or the absence (group 2; n = 113) of IAM. Clinical signs of patients, CT features, complications at surgery, and histological examinations were noted. Inter-observer agreement was assessed by using kappa statistics. Twenty-one patients presenting with CT features of IAM were diagnosed. An excellent inter-observer agreement (κ = 0.94) was assessed for the diagnosis of IAM. No significant statistical difference in the age distribution was observed between patients with IAM (mean age 55) and patients without (mean age 45) (p = 0.2232). No clinical sign showed a statistically significant association with the presence of IAM (p = 0.707) or with complication encountered at surgery (p = 0.180). Delay to CT examination was 5.4 days in patients presenting with CT features of IAM and of 1.7 days for patients presenting without (p = 0.0001). Conversely to acute appendicitis complicated by simple perforation (p = 0.153) or peri-appendicular abscess (p = 0.501), acute appendicitis presenting with IAM showed a statistically significant association with complications encountered at surgery (p = 0.0003) and the need for conversion to open surgery (p = 0.001). Performing CT in complicated acute appendicitis provides the diagnosis of IAM. Distinction of IAM appeared to be of clinical relevance, since immediate surgery in IAM was statistically associated with surgical complications and conversion to open surgery in our study.

Pre-irradiation of mouse mammary gland stimulates cancer cell migration and development of lung metastases.

BACKGROUND: In most patients with breast cancer, radiotherapy induces inflammation that is characterised by an increase of promigratory factors in healthy tissues surrounding the tumour. However, their role in the emergence of the migration phenotype and formation of metastases is still unclear. METHODS: A single mammary gland of BALB/c mice was irradiated with four doses of 6 Gy given at a 24-h interval. After the last session of irradiation, treated and control mammary glands were either collected for quantification of promigratory and proinflammatory factors or were implanted with fluorescent ubiquitination-based cell cycle indicator (FUCCI)-expressing mouse mammary cancer D2A1 cells. The migration of cancer cells in the mammary glands was monitored by optical imaging. On day 21, mammary tumours and lungs were collected for histology analyses and the quantification of metastases. RESULTS: Pre-irradiation of the mammary gland increased by 1.8-fold the migration of cancer cells, by 2-fold the quantity of circulating cancer cells and by 2.4-fold the number of lung metastases. These adverse effects were associated with the induction of interleukin-6 (IL-6) and cyclooxygenase-2 (COX-2). CONCLUSION: The emergence of the metastasis phenotype is believed to be associated with the accumulation of mutations in cancer cells. Our results suggest an alternative mechanism based on promigratory factors from irradiated mammary glands. In clinic, the efficiency of radiotherapy could be improved by anti-inflammatory agents that would prevent the stimulation of cancer cell migration induced by radiation.

Cirrhotic and malignant ascites: differential CT diagnosis.

OBJECTIVES: To assess the diagnostic accuracy of the different computed tomography (CT) signs for differentiating between malignant and cirrhotic ascites. MATERIALS AND METHODS: We performed a retrospective study of 102 CT scans in adults, distributed into two groups based on the cirrhotic or malignant etiology of their ascites. The CT signs studied were ascites volume and relative distribution between the greater peritoneal cavity (GPC) and the omental bursa (OB), the density of the ascites, the thickness of the gallbladder wall, the thickness of the parietal peritoneum and its degree of enhancement, and tethered-bowel sign. RESULTS: The CT signs associated with malignant ascites were: presence of fluid in the omental bursa (P=0.003), thickening of the peritoneum its degree of enhancement (P=0.005), increased density of the ascites (P=0.01), and loss of mobility of bowel loops in the ascites (P=0.001). There was no difference in gallbladder wall thickness between the two groups. CONCLUSION: The CT scan can play a role in diagnosing malignant ascites. We confirm the usefulness of the indirect signs composed of distribution of ascites fluid, thickening and enhancement of the parietal peritoneum, and loss of mobility of the bowel loops in the ascites.

Radiation-enhancement of MDA-MB-231 breast cancer cell invasion prevented by a cyclooxygenase-2 inhibitor.

BACKGROUND: Recent evidences support that radiation can promote the invasion of cancer cells. As interactions between cancer cells and surrounding stromal cells can have an important role in tumour progression, we determined whether an irradiation to fibroblasts can enhance the invasiveness of breast cancer cells. The role of cyclooxygenase-2 (COX-2), an inflammatory enzyme frequently induced by radiotherapy, was investigated. METHODS: Irradiated 3T3 fibroblasts were plated in the lower compartment of invasion chambers and used as chemoattractant for non-irradiated human breast cancer cell MDA-MB-231, which are oestrogen receptor negative (ER(-)) and the oestrogen receptor positive (ER(+)) MCF-7 cells. Stimulation of COX-2 expression in irradiated 3T3 cells was measured by a semi-quantitative qPCR and western blot. Capacity of the major product of COX-2, the prostaglandin E2 (PGE(2)), to stimulate the production of the matrix metalloproteinase-2 (MMP-2) and cancer cell invasion were assessed with a zymography gel and invasion chambers. RESULTS: Irradiation (5 Gy) of 3T3 fibroblasts increased COX-2 expression and enhanced by 5.8-fold the invasiveness of non-irradiated MDA-MB-231 cells, while their migration was not modified. Addition of the COX-2 inhibitor NS-398 completely prevented radiation-enhancement of cancer cell invasion. Further supporting the potential role of COX-2, addition of PGE(2) has increased cancer cell invasion and release of MMP-2 from the MDA-MB-231 cells. This effect of radiation was dependant on the expression of membrane type 1 (MT1)-MMP, which is required to activate the MMP-2, but was not associated with the ER status. Although irradiated fibroblasts stimulated the invasiveness of MDA-MB-231 ER(-) cells, no enhancement was measured with the ER(+) cell line MCF-7. CONCLUSIONS: Radiation-enhancement of breast cancer cell invasion induced by irradiated 3T3 fibroblasts is not dependant on the ER status, but rather the expression of MT1-MMP. This adverse effect of radiation can be prevented by a specific COX-2 inhibitor.

In vitro irradiation of basement membrane enhances the invasiveness of breast cancer cells.

Following removal of the primary breast tumour by conservative surgery, patients may still have additional malignant foci scattered throughout the breast. Radiation treatments are not designed to eliminate all these residual cancer cells. Rather, the radiation dose is calculated to optimise long-term results with minimal complications. In a tumour, cancer cells are surrounded by a basement membrane, which plays an important role in the regulation of gene expression. Using an invasion chamber, we have shown that irradiation before cell plating of a reconstituted basement membrane (Matrigel; Becton Dickinson, Bedford, MA, USA) increased the invasiveness of the breast cancer cells MDA-MB-231. This radiation enhancement of invasion was associated with the upregulation of the pro-invasive gene matrix metalloproteinase (MMP)-2. The expression of membrane type 1 matrix metalloproteinase (MT1-MMP) and tissue inhibitor of metalloproteinase-2 (TIMP), which are required to activate the MMP-2, were also increased. Confirming the role of MMP-2 and MT1-MMP, radiation enhancement of cancer cell invasion was prevented by an MMP-2 inhibitor and an anti-MT1-MMP antibody. This study also demonstrated that radiation can potentially enhance the invasion ability by inducing the release of pro-invasive factors stored in the Matrigel. Conversely, no enhancement of invasiveness was observed with the low metastatic cell line MCF-7. This lack of invasiveness correlated with the absence of the MMP-2 activator MT1-MMP in the MCF-7 cells. Radiotherapy is an efficient modality to treat breast cancer which could be further improved by inhibiting the pro-invasive gene upregulated by radiation.

Antioxidants to prevent bovine neutrophil-induced mammary epithelial cell damage.

Activated neutrophils are able to produce a large quantity of bactericidal molecules such as reactive oxygen species that have been associated with tissue damage in several inflammation models. The protective effects of antioxidants in a context of neutrophil-induced damage to mammary epithelial cells were first evaluated in vitro using a coculture model of activated bovine neutrophils and a bovine mammary epithelial cell line (MAC-T cells). Cell damage was determined by quantifying the release of lactate dehydrogenase by MAC-T cells in culture medium. Morphological observation of cells stained with acridine orange was used to visualize the extent of cell damage. When incubated with neutrophils activated by lipopolysaccharides and phorbol 12-myristate 13-acetate, MAC-T cells released large amounts of lactate dehydrogenase indicating significant cell damage. The addition of dimethylthiourea or bathocuproine disulfonic acid did not reduce the damage whereas catechin, deferoxamine or glutathione ethyl ester significantly reduced neutrophil-induced cytotoxicity in a dose-dependent manner. The effect of deferoxamine, an iron chelator, on the growth of Escherichia coli and the ability of bovine neutrophils to phagocytose these bacteria were then assessed in vitro. Our data showed that deferoxamine did not interfere with the phagocytic activity of neutrophils but inhibited growth of the bacteria. Overall, our results suggest that antioxidants may be effective tools for protecting mammary tissue against neutrophil-induced oxidative stress during bovine mastitis.

In vitro generation of peroxynitrite by 2- and 4-hydroxyestrogens in the presence of nitric oxide.

Estrogen metabolism is altered in most, if not all, breast cancer tumors. These alterations primarily lead to the formation of the catechol estrogen metabolites, 2- and 4-hydroxyestrogens, which can generate superoxide anion radicals (O(2)(*)(-)) through the redox cycling of semiquinone/quinone derivatives. In breast cancer cells, the activity of nitric oxide synthase is also frequently elevated, resulting in an increased level of exposure to nitric oxide ((*)NO). Since (*)NO rapidly reacts with O(2)(*)(-) to produce the peroxynitrite anion (ONOO(-)), this study was undertaken to determine whether ONOO(-) can be generated when 2- and 4-hydroxyestrogens are incubated in vitro with (*)NO donor compounds. Using dihydrorhodamine 123 as a specific probe for ONOO(-) formation, a ratio of 100 microM dipropylenetriamine NONOate (DPTA/NO) to 10 microM 4-hydroxyestradiol (4-OHE(2)) gave an optimal ONOO(-) production of 11.9 +/- 1.9 microM (mean +/- SD). Quantification of ONOO(-) was not modified by mannitol, supporting the idea that the hydroxyl radical was not involved. This production of ONOO(-) required the presence of the catechol structure of estrogen metabolites since all methoxyestrogens that were tested were inactive. Hydroxyestrogen metabolites derived from estradiol showed the same efficiency in producing ONOO(-) as those originating from estrone. With DPTA/NO, the 4-hydroxyestrogens generated 30-40% more ONOO(-) than the 2-hydroxyestrogens. Optimal production of ONOO(-) was assessed with DPTA/NO and diethylenetriamine NONOate (initial (*)NO generation rates of 0.76 and 0.08 microM min(-1), respectively). With faster (*)NO-releasing compounds, such as diethylamine NONOate and spermine NONOate, lower levels of ONOO(-) were detected. These data suggest that once the optimal concentration of (*)NO was obtained, the reaction between (*)NO and 4-OHE(2) was saturated. The excess of (*)NO would probably react with aqueous oxygen to form nitrite (NO(2)(-)). Since the third-order reaction rate for the reaction between 2(*)NO and O(2) is 2 x 10(6) M(-2) s(-1), it can therefore be suggested that the reaction between (*)NO and 4-OHE(2) occurs at a faster rate.

Thiols can either enhance or suppress DNA damage induction by catecholestrogens.

The estrogen metabolites catecholestrogens (or hydroxyestrogens) are involved in carcinogenesis and the development of resistance to methotrexate. This induction of drug resistance correlates with the relative efficiency of catecholestrogens in the generation of reactive oxygen species (ROS) and the induction of DNA strand breaks. Although antioxidants can neutralize ROS, the generation of these reactive species by catecholestrogens can be enhanced by electron donors like NADH. Therefore, this study was undertaken to determine the ability of different thiol agents (GSH, NAC, DTT, DHLA) to either inhibit or enhance the level of DNA damage induced by the H(2)O(2) generating system 4-hydroxyestradiol/Cu(II). Our results show that GSH, DTT, and DHLA inhibited the induction of the 4-hydroxyestradiol/Cu(II)-mediated DNA damage, with GSH showing the best potential. In contrast, the GSH precursor NAC at low concentrations was able to enhance the level of oxidative damage, as observed with NADH. NAC can reduce Cu(II) to Cu(I) producing the radical NAC&z.rad;, which can generate the superoxide anion. However, the importance of this pathway appears to be relatively minor since the addition of NAC to the 4-hydroxyestradiol/Cu(II) system generates about 15 times more DNA strand breaks than NAC and Cu(II) alone. We suggest that NAC can perpetuate the redox cycle between the quinone and the semiquinone forms of the catecholestrogens, thereby enhancing the production of ROS. In conclusion, this study demonstrates the crucial importance of the choice of antioxidant as potential therapy against the negative biological effects of estrogens.

Albumin-mediated regulation of cellular glutathione and nuclear factor kappa B activation.

Human serum albumin (HSA) is a cystine-rich serum protein taken up by many cells through receptor-mediated and fluid-phase endocytosis. We hypothesized that HSA may play a role in modulating cellular antioxidant redox signaling. Lung epithelial cells (A549), fibroblasts (HFL1), and blood lymphocytes had increased glutathione (GSH) levels after 8 h incubation with HSA. Similar GSH increases were observed with either plasma-derived or recombinant HSA. Serum depleted of HSA had no effect on cellular GSH. The GSH increase was also observed in normal murine lungs upon in vivo airway instillation of HSA. GSH enhancement was not related to the redox state of the free cysteine residue (Cys-34) on HSA, however, reduction of disulfide bonds in HSA inhibited the increase in cellular GSH. In addition, the albumin-mediated increase in GSH was inhibited by the vacuolar (H(+))-ATPase inhibitors, bafilomycin A(1) and concanamycin, as well as by the membrane pH-disrupting ionophore monensin, but not by 20 mM NH(4)Cl. The degree to which albumin increased GSH levels was sufficient to protect cells against H(2)O(2)-mediated cytotoxicity and to decrease TNF-alpha-mediated NF-kappaB activation. We conclude that albumin specifically modulates cellular GSH levels, an effect sufficient to protect cells against oxidant injury and regulate NF-kappaB activation.

Oestrogen metabolism in lymphangioleiomyomatosis: catechol-O-methyltransferase pathway is not involved.

BACKGROUND: Lymphangioleiomyomatosis (LAM) is an uncommon lung disease for which no effective method of treatment has been found. The predilection of LAM for premenopausal women has led to the assumption that hormonal factors play an important role in the pathogenesis of this disease. The aim of this study was to determine if women with LAM manifest alterations in the catechol-O-methyltransferase (COMT) pathway which is essential for preventing the generation of oestrogen derived reactive oxygen species (ROS). METHODS: Blood samples were collected from 15 women with LAM and compared with appropriate controls. The distribution of high and low activity alleles of COMT was determined with a PCR based RFLP assay. The enzymatic activity of COMT was measured in each sample and the potential presence of a circulating inhibitor of COMT was determined. Since an alteration in the COMT pathway could increase the oxidative stress, the plasma concentration of malondialdehyde (MDA), a secondary product generated from lipid peroxidation, has been used as an internal marker. RESULTS: The distribution of high and low activity alleles of COMT (named COMT(HH), COMT(LL), and COMT(HL)) was similar in the two groups with proportions of 40%, 7%, and 53%, respectively, in the women with LAM and 38%, 6%, and 56% in the control subjects. The mean (SD) COMT activity was 24.2 (12.3) pmol/min/mg protein in women with LAM and 24.1 (6.3) pmol/min/mg protein in the control group. Incubation of plasma from women in the two groups with a preparation of commercial COMT showed that no detectable COMT inhibitor was present. The plasma concentration of MDA in the women with LAM was also not significantly different from control subjects. CONCLUSIONS: This study shows that there are no significant alterations in the COMT pathway of women with LAM. It is therefore unlikely that alterations in oestrogen mediated cell signalling pathways are mediated by oxidants derived from an excess of catecholoestrogens in LAM.

DNA damage induced by catecholestrogens in the presence of copper (II): generation of reactive oxygen species and enhancement by NADH.

Certain estrogen metabolites are involved in carcinogenesis and the development of resistance to methotrexate (MTX). In this study, we determined whether these well-established biological effects correlate with the relative efficiency of several estrogen metabolites to induce DNA strand breaks in the presence of copper, and investigated the potential enhancing effect of reduced nicotinamide adenine dinucleotide (NADH). DNA strand breaks induced by estradiol metabolites were measured by the conversion of supercoiled phage phiX-174 RF1 DNA to open circular and linear forms. The most active catecholestrogens were the 4-hydroxy derivatives, which produced about 2.5 times more DNA double strand breaks than the 2-hydroxy derivatives, while estradiol and 16alpha-hydroxyestrone were inactive. In addition, our results show that 4-hydroxyestradiol (4-OHE2) at physiological concentrations was capable of exhibiting DNA cleaving activity. The formation of these catecholestrogen-induced DNA strand breaks was associated with the utilization of oxygen and the generation of H2O2, because catalase inhibited the DNA cleaving activity of 4-OHE2. Interestingly, we also observed that NADH enhanced the induction of DNA strands breaks by 4-OHE2/Cu(II), probably by perpetuating the redox cycle between the quinone and the semiquinone forms of the catecholestrogen. In conclusion, this study demonstrated that the relative efficiency of 2-, and 4-hydroxyestrogen in carcinogenesis and for the enhancement of MTX resistance correlates with their relative capability to induce DNA strand breaks. In order to inhibit these estrogen-mediated biological effects, it may be important to develop different strategies to block the production of reactive oxygen species by the catecholestrogen-redox cycle.

Induction by estrogens of methotrexate resistance in MCF-7 breast cancer cells.

Development of drug resistance is a major factor that limits the effectiveness of chemotherapy treatments. In this study, we determined whether estradiol or its metabolites 2-, 4- and 16alpha-hydroxyestrone could enhance the development of methotrexate resistance in the breast carcinoma cell line, MCF-7. Cells were incubated with the estrogens at a concentration of 10(-8) M for 12 cell doublings and enhancement of methotrexate resistance was measured with the Luria-Delbrück assay. The most efficient estrogens were the 4-hydroxyestrone and 16alpha-hydroxyestrone, which both stimulated methotrexate resistance by 88-fold as compared with the control without estrogen. 2-Hydroxyestrone had an enhancement factor of 33-fold, whereas estradiol showed a slight effect with an enhancement factor of 3.2-fold. To determine whether the estrogen receptor was involved in the development of resistance, expression of the pS2 gene, which contains an estrogen-responsive element, was measured. Both estradiol and 16alpha-hydroxyestrone stimulated expression of the pS2 gene. In contrast, 2- and 4-hydroxyestrone did not increase the level of pS2 mRNA. This suggests that tumors classified as estrogen receptor negative could also develop methotrexate resistance as the result of exposure to estrogens. The status of the tumor suppressor gene p53 was analyzed in methotrexate sensitive and resistant clones. In all the methotrexate resistant clones analyzed, the western blots indicated that the p53 protein was still present and transcriptionally competent, as measured by its capacity to stimulate transcription of the p21waf1/cip1 gene following UVB irradiation. However, the basal level of p53 was higher in resistant clones and addition of 2- or 4-hydroxyestrone increased p53 to levels equivalent to those observed following UVB irradiation. However, this induction of p53 accumulation by estrogens failed to stimulate the transcription of p21waf1/cip1, which indicates that a transcriptionally inactive form of p53 accumulated in methotrexate resistant cells.

Enhancement of genomic instability by 17beta-estradiol in minisatellite sequences of X-ray-transformed mouse 10T1/2 cells.

The female hormone 17beta-estradiol is involved in the development of breast cancer, an effect usually attributed to its capacity to stimulate the replication of preneoplastic and malignant cells. In this study, we report that 17beta-estradiol enhances the onset of genomic rearrangements, a type of genomic instability, in minisatellite sequences of malignant 10T1/2 mouse cells. Two malignant clones, X-ray-9 and F-17a, previously transformed in vitro by X-rays (600 cGys), and two non-transformed 10T1/2 mouse cell subclones (10T1/2b and 10T1/2c) were divided into two groups. The first group was incubated in the presence of 10(-5) M of 17beta-estradiol (dissolved in ethanol) for 5 days, while the second group was incubated for the same period in culture media containing 0.1% of ethanol. After the incubation both groups of cells were then subcloned, and their DNA was extracted and analyzed with the DNA fingerprinting assay using the probe M (core sequence: 5'-AGGC). A high frequency of genomic rearrangements was observed in the transformed subclones treated with 17beta-estradiol. Nine deletions or additions in minisatellite alleles were observed in six F-17a subclones, while 28 of those genomic rearrangements were found in the 12 X-ray-9 malignant subclones. On the other hand, for the non-transformed 10T1/2b and 10T1/2c cells, no genomic rearrangements were induced by the hormone. After the withdrawal of 17beta-estradiol from the transformed clone X-ray-9, no new genomic rearrangements were detected; while a second incubation with the hormone induced new deletions or additions in minisatellite alleles. This preferential enhancement of genomic instability in malignant 10T1/2 mouse cells suggests that 17beta-estradiol may accelerate the accumulation of mutations, and therefore may represent a mechanism by which the female hormone contributes to breast cancer development.

In vitro reversion of transformed phenotype in mouse C3H/10T1/2 cells.

Spontaneous tumor regression is still one of the most puzzling events in human cancer. A cell culture model of malignant transformation designed to permit the study of this phenomenon in vitro was applied to examine reversion and re-expression of the transformed phenotype in two X-ray transformed mouse 10T1/2 cell clones. By alternating cell passages at low and high seeding density, the expression of cell contact inhibition and tumorigenic capacity were both reverted and restored. Growth of non-transformed wild-type cells was not affected by seeding density. This reversion of the transformed phenotype was associated with a modification in genomic 5-methylcytosine content. Initially, the transformed clones were hypomethylated, as occurs in most human tumors. After only four passages at low seeding density, the phenotype was reverted to that of non-transformed 10T1/2 cells and genomic 5-methylcytosine content was significantly increased to levels measured in non-transformed C3H/10T1/2 mouse cells. Thus, hypomethylation induced by ionizing radiation was not a permanent feature of malignantly transformed 10T1/2 cells. Although genomic 5-methylcytosine content returned to normal levels during low density passaging, the methylation pattern of the c-myc gene specifically was not associated with cell passages either at low or high seeding density. In an attempt to identify genes involved in this process, expression of the tumor suppressor gene p53 was measured. Western blot analysis failed to detect any correlation between expression of p53 protein and reversion of the transformed phenotype. The results of this study indicate that the transformed phenotype is not permanently associated with the malignant transformation of C3H/1OT1/2 cells, and can be modulated by growth conditions in vitro. We propose that modulation of genomic 5-methylcytosine levels may be involved in this process.

In vivo enhancement of genomic instability in minisatellite sequences of mouse C3H/10T1/2 cells transformed in vitro by X-rays.

The level of genomic instability was determined during tumor development in vivo. Genomic rearrangements, a marker of genomic instability, was measured in mouse C3H/10T1/2 cells transformed in vitro by X-rays with a DNA fingerprinting assay. Three transformed clones isolated from type III foci were divided into two groups. Cells from the first group were injected s.c. into syngeneic and nonimmunosuppressed C3H mice. After 3 to 5 months, the tumors were excised, and the neoplastic cells were isolated and subcloned. Cells from the second group were incubated in vitro for 25 passages (about 6 months) to approximate the number of cell divisions occurring in the tumor, and then they were subcloned. DNA was extracted from subclones grown in vitro and in vivo and analyzed with the DNA fingerprinting assay. A high frequency of genomic rearrangements (50-100%) was found in subclones derived from tumors that arose in vivo, whereas the frequency was very low (< 10%) among subclones passaged in vitro, suggesting that genetic instability may be enhanced by factors present in the C3H mouse. In one clone (F-17) genomic instability appeared to be activated and down regulated. The high frequency of instability found in tumor cell subclones did not appear to result from an in vivo selection of a more tumorigenic subpopulation of cells present in the original clone prior to injection in the animal. This enhancement of genomic instability occurring in vivo could be required to complete the process of transformation to tumorigenicity and allow the neoplastic cells to adapt to a new environment.