Hormone-dependent human tumors express the fms gene-product.

Human mammary and thyroid tumors as well as peripheral blood cells of the same patients were examined for the amplification of oncogene fins and the expression of its product. Of the 7 mammary tumors analyzed, amplification of fms was observed in 6 (86%) mammary tumor DNAs, while no amplification was seen in 5 thyroid tumors and 7 peripheral blood cell DNAs tested. None of the mammary and thyroid tumors showed any rearrangement of the fms gene. Investigations of the expression of the product of fms in various human tumors were carried out following the method of immunohistochemical staining in which polyclonal antibody to the fms gene product was used. The incidence of the expression of fms gene product in the tumors of mammary and thyroid glands, prostate and ovary, the hormone-dependent organs, was 38-97%, whereas fms protein was found to be present only in 20 and 42% of the tumors from hormone-independent organs, the brain and the bladder. Expression of the fms gene product was not detectable in normal tissue surrounding the tumor tissue in any of the cases examined. These results suggest that the expression of the product of fms may be associated with the development of some tumors of hormone responsive organs, especially the breast and thus the fms gene product may be a valuable marker for human mammary tumors.

Overexpression of the tumor-suppressor gene p53 in human ovarian tumor-tissues and its correlation with clinical stage.

An extensive series of histological sections from various types of benign, borderline, and malignant human ovarian tumors were examined for expression of the p53 protein by immunohistochemical staining, using a new anti-p53 mouse monoclonal antibody. Positive staining for p53 was detected in 34% of all malignant ovarian cancer tissues. A high rate of positivity was observed in serous cystadenocarcinoma (59.7%). None of the borderline or benign tumors showed any reaction. No relation was observed between clinical stage, and the frequency of p53 positivity. Mutation of the p53 gene with overexpression of the mutant protein appears to be a specific genetic change in human ovarian cancer.

Resveratrol inhibits human breast cancer cell growth and may mitigate the effect of linoleic acid, a potent breast cancer cell stimulator.

Resveratrol is a naturally occurring product found in grapes and wine. The effect of synthetic resveratrol on the growth of estrogen receptor (ER)-positive (KPL-1 and MCF-7) and -negative (MKL-F) human breast cancer cell lines was examined. Resveratrol at low concentrations caused cell proliferation in ER-positive lines (KPL-1, < or = 22 microM; MCF-7, < or = 4 microM) whereas at high concentrations (> or = 44 microM) it caused suppression of cell growth in all three cell lines examined. Growth suppression was due to apoptosis as seen by the appearance of a sub-G1 fraction. The apoptosis cascade up-regulated Bax and Bak protein, down-regulated Bcl-xL protein, and activated caspase-3. Resveratrol (52-74 microM) antagonized the effect of linoleic acid, a potent breast cancer cell stimulator, and suppressed the growth of both ER-positive and -negative cell lines. Thus, resveratrol could be a promising anticancer agent for both hormone-dependent and hormone-independent breast cancers, and may mitigate the growth stimulatory effect of linoleic acid in the Western-style diet.

Effects of genistein and synergistic action in combination with eicosapentaenoic acid on the growth of breast cancer cell lines.

Genistein, a prominent isoflavone in soy products, produced dose- and time-dependent in vitro growth inhibition at high concentrations (at least 185 microM) with an IC50 of 7.0-274.2 microM after 72 h incubation in four breast cancer cell lines (DD-762, Sm-MT, MCF-7 and MDA-MB-231) and one breast epithelial cell line (HBL- 100) of human and animal origin; it stimulated estrogen-receptor-positive MCF-7 cells at low concentrations (3.7 nM-37 microM). Genistein-exposed cells underwent apoptosis, confirmed by G2/M arrest followed by the appearance of a sub-G1 fraction in cell-cycle progression, and by a characteristic cell ultrastructure. The apoptosis cascade was due to up-regulation of Bax protein, down-regulation of Bcl-XL protein, and activation of caspase-3. Genistein acted in synergism with eicosapentaenoic acid (EPA), a fish oil component, on human breast cancer MCF-7 cells (genistein > 93.2 microM and EPA > 210.9 microM) and on MDA-MB-231 cells (genistein > 176.1 microM and EPA > 609.3 microM). Dietary intake of genistein in combination with EPA may be beneficial for breast cancer control.

Cycloprodigiosin hydrochloride, a H+/Cl- symporter, induces apoptosis in human breast cancer cell lines.

The effect of cycloprodigiosin hydrochloride (cPrG.HCl), a H+/Cl- symporter, on five human breast cancer cell lines (KPL-1, T-47D, MCF-7, MKL-F, and MDA-MB-231), a human breast epithelial cell line (HBL-100), and a human fibroblast cell line (WI-38-40) was examined. cPrG.HCl inhibited the growth of all five breast cancer cell lines (IC50: 0.46-0.62 microM) and slightly inhibited HBL-100 and WI-38-40 cell growth (IC50: 1.75 microM and 2.26 microM respectively). cPrG.HCl treatment in KPL-1 cells increased the pH of acidic organelles, decreased intracellular pH, and caused apoptosis, which was confirmed by the appearance of a sub-G1 population by flow cytometry and DNA fragmentation. In addition, cPrG.HCl-induced apoptosis was strongly suppressed by imidazole, a cell-permeable base, suggesting that intracellular acidification was essential for the apoptosis. Further, cPrG.HCl treatment up-regulated Bax and Bak expression, down-regulated Bcl-2 expression, and activated caspase-3. Therefore, the intracellular acidification by cPrG.HCl treatment suppressed the growth of human breast cancer cell lines by inducing apoptosis.

Novel cell lines established from a human myxoid malignant fibrous histiocytoma arising in the uterus.

Two cell lines (Nara-H and Nara-F) with different phenotypes were established from a myxoid MFH of the uterus. In vitro, Nara-F grew in sheets showing a storiform arrangement and Nara-H in raised colonies. Although tumors generated in nude mice shared similar morphological features of abundant myxoid tumor in Nara-H and -F, the pleomorphic component was conspicuous in Nara-F. Both cell lines produced hyaluronic-acid but CD44 was expressed only in Nara-H. Estrogen receptor alpha (ER alpha) and progesterone receptor (PgR) were detected in Nara-H. Nara-F was positive for ER beta and PgR. Among hormonal agents, the response to the anti-estrogen tamoxifen was more sensitive than progesterone agents. This report illustrates the characteristics of these newly established cell lines, and presents the possibility of an adjuvant hormonal therapy for MFH.

Novel germ-cell tumor-cell line established from a testicular feminization syndrome.

We analyzed a novel cell line established from a testicular feminization syndrome. The original tumor was an endodermal sinus tumor with seminoma. The doubling time, the saturation density and the mitotic index were 36.1 hours, 3.65x10(4) cells/cm2 and 4.3%, respectively. The mode number of chromosomes was 102. Results of the MTT assay showed that the cell line had an IC50/PPC of less than 0.1 for methotrexate and actinomycin D. The effects of methotrexate were schedule dependent; the effects of actinomycin D were both schedule and dose dependent.

Study of angiotensin II-induced hypertensive chemotherapy for ovarian cancer in rats.

We investigated the usefulness of angiotensin II (AT II)-induced hypertensive chemotherapy for adenocarcinoma of the ovary, which was induced with dimethylbenzantracene (DMBA) in rats. Ovaries with DMBA-induced cancer had poor blood flow, while normal ovaries had abundant brood flow when blood pressure was normal. The brood flow of the normal ovaries was suppressed by AT II-induced hypertension, while the blood flow in ovarian cancer was more than doubled. Tissue levels of the anticancer drug cisplatin (CDDP) were almost the same in the normal ovary in the presence or absence of induced hypertension. However, the CDDP level in ovarian cancer tissue was significantly higher with AT II-induced hypertension than with normotension. Comparison of the ratio of the enlarged tumor to the primary tumor and the histopathologic antitumor effects, showed that AT II-induced hypertension treatment with CDDP was highly effective. Angiotensin II-induced hypertensive chemotherapy for adenocarcinoma of the ovary in rat was highly effective in comparison with normotension.

Relation between CA125 levels and second-look operation for ovarian cancer.

We investigated the relationship between the serum level of CA125 before a second-look operation (SLO) and SLO findings in 196 patients with adenocarcinoma of the ovary. SLO findings were positive in 38 (19.4%) of 196 patients. The positive rate tended to increase with the clinical stage, but SLO findings were positive even in a patient with stage Ia disease. The pre-SLO serum level of CA125 was positive in 11 patients before SLO, SLO findings were positive in 8 of these 11 patients. The highest diagnostic accuracy (37.9%) for the pre-SLO serum level of CA125 was obtained at a cut-off value of 11 U/ml. Our findings suggest that a positive pre-SLO serum level of CA125 does not necessarily indicate tumor positivity. In addition, we suggest that the cut-off value for prediction of SLO findings should be below 35 U/ml, which is a commonly used cut-off value.

Fertility-preserving treatment for advanced malignant germ cell tumors of the ovary.

We retrospectively studied 26 patients with stage Ic or greater malignant germ cell tumors of the ovary. Sixteen of the 26 underwent fertility-preserving treatment and 10 underwent total hystero-oophorectomy. There were 3 deaths in each group, but none among the stage Ic cases. The survival curves for stage II to IV disease did not significantly differ in the two groups. Seven patients in the fertility-preserving group were married, and had children. All patients in the hystero-oophorectomy group remained single except for one who was married before surgery. These results suggest that fertility-preserving treatment should be offered even in advanced cases.

Expression and localization of matrix metalloproteinases and tissue inhibitors of metalloproteinases as a prognostic factor in advanced colorectal carcinomas.

To clarify the usefulness of matrix metallo-proteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) as prognostic factors in advanced colorectal carcinoma, the immunohistochemical expressions of MMP-1, MMP-2, MMP-3, MMP-9, TIMP-1 and TIMP-2 were examined. Specimens were selected from 67 consecutive patients undergoing surgery for advanced colorectal carcinoma. The patterns of expression were compared with the prognoses of the patients. The patients with TIMP-2 expression in stroma adjacent to the tumor mass had better prognoses than those of the patients who had no TIMP-2 expression in normal stroma adjacent to the tumor (p<0.05), which probably acted as a block of cancer cell invasion. However, the expression of MMP-2, presumably acting as an antagonist to TIMP-2 was not related to the prognosis, and the MMP-1, MMP-2, MMP-3, MMP-9 and TIMP-1 expressions were not related to any clinicopathological factors examined.

Macrophage colony-stimulating factor as a marker of ovarian carcinoma.

Macrophage colony stimulating factor (M-CSF) is a cytokine which stimulates the proliferation and differentiation of phagocytic cells. We evaluated the usefulness of M-CSF as a serum tumor marker for ovarian malignancies and also assessed M-CSF production by tumor cells and the role of an autocrine system in such M-CSF production. The findings obtained were as follows: i) Serum M-CSF was a useful marker for malignant ovarian tumors. ii) M-CSF was a marker for both epithelial stromal tumors and for germ cell tumors. It was also a marker for dysgerminoma, for which no specific tumor marker is currently available. iii) The value of combined assays employing M-CSF was confirmed. iv) M-CSF production was demonstrated in various malignant ovarian tumor cell lines, but the presence of an autocrine system for M-CSF was not confirmed.

Ovarian tumor complicated by a colo-ovarian fistula.

Although a fistula is a rare complication of ovarian tumor, we encountered four patients with cole-ovarian fistulas in three years. The first case was demonstrated by following barium enema by the extravasation of barium from the sigmoid colon, and by a gas-containing lesion in the tumor observed on computed tomography. Mature cystic teratoma was the pathological diagnosis in two cases. A mixed germ cell tumor and a serous cystadenoma of low malignant potential were diagnosed in the other two cases. The etiology and differential diagnosis of cole-ovarian fistula are reviewed.

Case of leiomyomatosis peritonealis disseminata treated with a gn-rh analog.

We report a rare case of leiomyomatosis peritonealis disseminata (LPD) which we attempted to treat by administering the Gn-RH superagonist, buserelin. A 48-year-old woman presented with a 6-month history of hypermenorrhea. Hysterectomy revealed a benign leiomyoma. Light and electron microscopic studies revealed that the tumors were composed of well- or poorly-differentiated smooth muscle cells. Their clinical appearance together with the mitotic figures found in tumor sections suggested an uncertain malignant potential. In the immunohistochemical study of intermediate filaments, antidesmin and anti-actin antibody reacted with tumor cells. Tumor re-growth in the pelvic cavity led to a second operation. The administration of the Gn-RH analogue, 900 mu g daily for two months, decreased the tumor size. The average final volume was 68.7% of the original. However, the tumors recurred during the next four weeks. The combined administration of the Gn-RH analogue and etoposide (25 mg x 2/day) did not prevent recurrence after a third operation. The serum level of immune-suppressive acid protein (IAP) was useful in monitoring the clinical course of this patient, finally diagnosed as having LPD.

In-vitro cytotoxicity in ovarian-cancer of low-dose continuous exposure to microtubule inhibitors (Paclitaxel taxotere) combined with Cisplatin.

We present our data on the cytotoxicity of microtubule inhibitors (paclitaxel and taxotere) and their mode of action against cisplatin-sensitive or -resistant ovarian cancer cell lines in vitro. To try to establish an optimal administration schedule in clinical use, the cytotoxicity of microtubule inhibitors alone and in sequential combination with cisplatin was investigated. With microtubules alone, a marked dose- and schedule-dependent growth inhibition was demonstrated in lower concentrations and only schedule-dependency in higher concentrations. The cytotoxicity of taxotere was approximately 0.8- to 132-fold that of paclitaxel. No cross resistance to cisplatin was observed. In the relation between assay AUC (area under the concentration curve) and growth inhibition, increasing AUC by dose escalation seemed not to be advantageous. In a combination with cisplatin, the treatment of microtubules over 48 h followed by cisplatin administration demonstrated the most effective cytotoxicity in cisplatin-resistant cell line.

Telomere length, telomerase activity and telomerase RNA expression during mouse mammary tumor progression.

To investigate the roles of telomere length (mean length of the terminal restriction fragments; TRFs), telomerase activity (TA) and telomerase RNA (mTR) expression in relation to mouse mammary tumor progression, we examined a pregnancy-dependent mouse mammary tumor line (TPDMT-4) and its four autonomous sublines (T4-OI320: non-metastatic; and T4-OI165, -OI96, and -OI145: artificial metastatic) of DDD/1 mouse origin, and an autonomous growing mammary tumor (JYG-MC) showing spontaneous lung metastasis developed in BALB/c mice infected with a Chinese feral mice (Sub-Jyg)-derived mouse mammary tumor virus (JYG-MTV). Compared with normal (pregnant) mammary tissue, the TA was elevated in the TPDMT-4 tumor and in the non-metastatic subline tumor (T4-OI320) (x10 fold, respectively), and was further increased (x13-15 fold) in parallel with the acquisition of metastatic potential (T4-OI165, -OI96, and -OI145). The mTR level was upregulated (x2.7-2.8 fold) in all autonomous growing tumors compared to the normal counter-part, but not in TPDMT-4. The TRF was shorter in accord with tumor progression (normal mammary tissue, 48 kb; TPDMT-4, 45 kb; T4-OI320, 37 kb; T4-OI165, -OI96 and -OI145, mean 37.7 kb; and JYG-MC, 21 kb). These results suggest that the activation of TA occurs as an early event at the stage of hormone-dependent tumorigenesis, followed by the up-regulation of mTR expression in accordance with the acquisition of autonomous growth, and then further activation of TA occurs when the tumor acquires metastatic potential. The TRF shortening was in parallel with the tumor progression.

Change in telomerase activity during human colorectal carcinogenesis.

Telomerase activities in endoscopically resected colorectal adenomas, surgically resected colorectal cancers and adjacent normal colonic mucosa were examined semiquantitatively by a polymerase chain reaction-based telomeric repeat amplification protocol (TRAP) assay. All normal mucosa (n = 15) presented weak telomerase activity (mean +/- SE: 0.99 +/- 0.00). When the value of 1.00 was arbitrary given to the mean activity of normal mucosa, the telomerase activity in the adenomas (n = 14) was up-regulated (2.01 +/- 0.22) relative to the normal mucosa. The telomerase activity in the high-grade atypia (severe atypia and carcinoma in situ) (2.58 +/- 0.34) was significantly higher than that in the low-grade (mild and moderate) atypia (1.59 +/- 0.18) (P < 0.05), and the adenomas 10 mm or more in diameter presented significantly higher telomerase activity (2.56 +/- 0.09) than compared to the smaller ones (1.44 +/- 0.17) (p < 0.05). Carcinomas (n = 20), showed a telomerase activity that varied from 0.97 to 16.93, which was than the greater mean telomerase activity (6.96 +/- 1.25) noted in the adenomas. The telomerase activity in the carcinomas tended to be higher in the larger (> or = 4 cm), histologically less-differentiated (moderately differentiated), late-stage (Dukes C + D), and nodal metastatic tumors, suggestive of unfavorable prognosis. These results suggests that the weak telomerase activity in normal colonic mucosa is gradually activated during the course of colorectal carcinogenesis.

Telomere length, telomerase activity and telomerase RNA expression in human esophageal cancer cells: correlation with cell proliferation, differentiation and chemosensitivity to anticancer drugs.

The telomere and the enzyme telomerase in esophageal cancer have been poorly investigated. We present here aspects of the telomere and telomerase in esophageal cancer in relation to cell proliferation, differentiation and chemosensitivity to anticancer drugs. The telomere length (mean length of telomere restriction fragments; TRF), telomerase activity (TA), and human telomerase RNA (hTR) expression in a panel of 13 human esophageal cancer cell lines, squamous in origin, was examined by Southern blotting, the telomeric repeat amplification protocol (TRAP), and reverse transcriptase-polymerase chain reaction (RT-PCR), respectively. Cell proliferation expressed by the doubling time, cell differentiation determined by the keratin 13 and/or 14 expression, and chemosensitivity to cisplatin (CDDP) and 5-fluorouracil (5-FU) were compared with telomere-related factors. TRF shortening, the up-regulation of TA, and hTR expression was seen in all 13 cell lines. The TA correlated positively with the telomere length and negatively with the hTR expression. The doubling times of the cell lines and the telomere-related factors did not show any significant relation. The TA in the keratin 13/14-negative cell lines was significantly higher than that of the keratin 13-positive cell lines. The cells with short telomere tended to be resistant to CDDP whereas the cells with higher TA tended to be more sensitive to CDDP; 5-FU showed no relation to any telomere-related factors. Therefore, the activation of TA in esophageal squamous cell carcinoma is regulated by cell differentiation but not by cell proliferation, cells with high TA are more sensitive to CDDP, and cells with short telomere require a CDDP dose escalation.

Correlation of chemosensitivity to anticancer drugs and telomere length, telomerase activity and telomerase RNA expression in human ovarian cancer cells.

BACKGROUND: We examined the correlations among telomere length (TRF), telomerase activity (TA) and the steady-state level of telomerase RNA expression (hTR) of human ovarian cancer cells with different phenotypes and investigated whether the cells' sensitivities to anticancer agents correlate with TRF, TA or hTR. MATERIALS AND METHODS: The TRF, TA and hTR of 11 human ovarian cancer cell lines and 2 cisplatin-resistant ovarian cancer cell lines were determined by genomic Southern blotting, a telomeric repeat amplification protocol and reverse transcription-polymerase chain reaction. The chemosensitivities of the cell lines to cisplatin (CDDP), paclitaxel (TAX), etoposide (ETO), CPT-11 (CPT), cyclophosphamide (CYC), ifomide (IFO) and doxorubicin (DOX) were decided as IC50 values by calorimetric assay. RESULTS: All 11 cell lines presented shorter mean TRFs (5.0 kb) than normal control tissue (8.0 kb); 10 cell lines presented a 3.2-fold higher mean TA than the control and all 11 cell lines expressed hTR. Quantitatively, the steady-state levels of hTR correlated with the TRF (p < 0.05). Significant positive correlations between hTR and CDDP sensitivities (at 24 hours of exposure), ETO (72 hours), CPT (48 hours) and CYC/IFO (24-72 hours) were observed. The same was true for TRF and the CDDP sensitivities (at 24 hours). TAX and DOX did not have any impact on these factors. The TRF, TA and hTR values in the two CDDP-resistant cell lines were generally reduced, compared to their parent cell lines. CONCLUSIONS: Alkylating agents (CDDP, CYC and IFO) and topoisomerase inhibitors (ETO, CPT) may have the potential to influence the structural alteration of hTRs and telomeres and thus, the down-regulation of the TA in ovarian cancer cells.

Dietary effects of fatty acids on growth and metastasis of KPL-1 human breast cancer cells in vivo and in vitro.

To evaluate the effects of dietary fats on breast cancer growth and metastasis, KPL-1 human breast carcinoma cells which have a propensity for axillary lymph node metastasis when inoculated into the thoracic mammary fat pad of female nude mice were examined. The mice were fed one of three semipurified diets containing 9.5% eicosapentaenoic acid plus 0.5% linoleic acid (EPA diet), 10% linoleic acid (LA diet), or 9.5% palmitic acid plus 0.5% linoleic acid (PA diet), or commercial laboratory chow containing 8.5% fat of which 4.1% was LA, 1.1% was PA, 0.06% was EPA, and 3.24% was other (Standard diet) starting 19 days before tumor cell inoculation and continuing until the end of the experiment (43 days after tumor cell inoculation). The tumor growth was faster and at a higher incidence in the mice fed the LA diet, and much slower and at a lower incidence in the EPA diet group compared with the mice fed the PA or Standard diet; the two separate experiment demonstrated identical results. The differences in tumor weight between the LA and PA groups and between the PA and EPA groups were significant (P < 0.05, respectively) at the termination of the experiment; the differences were due to different tumor cell proliferation rates. In an in vitro MTT assay, fatty acids showed direct stimulatory or inhibitory effects on the KPL-1 cells. Lymph node metastasis was seen in the LA and Standard diet groups, whereas it was not seen in the PA or EPA groups. The body weights were significantly lighter in the LA and EPA groups compared with the PA and Standard diet groups (P < 0.05, respectively). The results indicate that the EPA diet produced a reduction in tumor cell growth and metastasis whereas the LA diet had an enhancing effect on these parameters; dietary fatty acids may thus have a direct role in the growth and metastasis of human breast carcinoma independent of their systemic effects.