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Six non-small cell lung cancer (NSCLC) cell lines (A-549, Ca-Lu-6, SK-Lu-1, Ca-Lu-1, SK-Mes-1 and LX-1) were studied to assess the presence of multiple concomitant alterations of different oncogenes (K-ras, bcl-2) and tumor suppressor genes (p53, Rb) in NSCLC. K-ras (exon 1) and p53 (exons 5-8) gene mutations were determined via a PCR-based-DGGE (Denaturing Gradient Gel Electro-phoresis) and by sequencing approach. Different mutations were found in the Ist exon of K-ras gene in 5 of 6 cell lines examined. Five of six cell lines contained K-ras mutations at codon 12 (A-549, SK-Lu-1, LX-1) or codon 13 (SK-Mes-1, Ca-Lu-1). In addition, 5 of 6 cell lines showed p53 mutations of exon 8 (SK-Mes-1, Ca-Lu-1 cod. 280; LX-1 cod. 273) or exon 6 (Ca-Lu-6 cod. 196; SK-Lu-1 cod. 193). In 4 of these cell lines, p53 protein nuclear expression was also confirmed with DO-7 mAb immunocytochemistry. Expression of cytoplasmic bcl-2 protein, by anti-bcl-2 mAb flow cytometric analysis, was found in A-549, Ca-Lu-1, SK-Lu-1, SK-Mes-1 cell lines. In contrast, RT-PCR analysis of Rb gene could not identify any change in the cell lines examined. In conclusion, most NSCLC cell lines tested displayed concomitant multiple oncogene/tumor suppressor gene alterations.

According to the conflicting growth signal model, cells that are driven to proliferate by certain oncogenes undergo apoptosis but not growth arrest upon withdrawal of growth factors. However, we found that the majority of human cancer cell lines continued to proliferate and did not undergo apoptosis following serum withdrawal. As an exeption, wild-type (wt) p53-expressing HCT116 human colon cancer cells underwent apoptosis within 24-36 h of serum deprivation. p53 degradation in human papilloma virus EG-expressing HCT116 cells led to enhanced survival that was not due to growth arrest. These results are consistent with a role for p53 in starvation-induced death in HCT116 cells. However, other cell lines did not undergo apoptosis despite their expression of wt p53. Thus, H460 cells (wt p53) were resistant to starvation-induced death but introduction of the adenovirus EIA oncoprotein induced p53 and also increased sensitivity to serum withdrawal. p53 was not stabilized by E1A and resistance to starvation-induced cell death was observed in E6-expressing H460 cells. These results suggest that although p53 contributes to starvation-induced apoptosis in sensitive (HCT116 and E1A-expressing H460) cancer cell lines, most cancer cells survived despite the presence of wt p53. We conclude that naturally selected human cancer cell lines suppress apoptosis due to conflicting growth signals.

Previous studies from this laboratory have shown that bioflavonoids including luteolin and quercetin possess antiestrogenic activity in the female reproductive tract and appear to elicit this activity by interaction with type II [H-3]estradiol binding sites in the cell nucleus. Studies by a number of laboratories including our own have shown that type II sites are present in the rodent prostate gland and therefore, we suspected that administration of bioflavonoids such as luteolin may antagonize prostate growth through type LT site binding interactions as well. The studies presented in this report demonstrate that the mouse prostatic tissue does contain nuclear type II sites and that oral administration of luteolin for 14 days results in a significant (p<0.01) reduction in prostatic weight in intact male mice without significant effects on the seminal vesicular, testis or body weights of these animals. These results suggest that luteolin is a prostate specific antagonist under these experimental conditions. In vitro studies with LNCaP and PC-3 human prostate cancer cell lines demonstrated that luteolin treatment resulted in a dose dependent inhibition of prostate cancer cell proliferation which was maximum 4-8 days following treatment. Whole cell binding studies demonstrated that both LNCaP and PC-3 cells contained very high concentrations of type II [H-3]estradiol binding sites (>200,000 sites/cell) relative to levels previously reported for other tissues and cells and luteolin was capable of interacting with these sites in prostate cancer cells. In fact, there was a direct correlation between the type II site occupancy by luteolin and the inhibition of LNCaP or PC-3 prostatic cancer cell proliferation by this bioflavonoid. Flow cytometric analysis revealed that luteolin treatment caused an accumulation of LNCaP cells in G(2)/M (p<0.01) and reduced the fractions of LNCaP cells in S-phase undergoing apoptosis (p<0.01). Similarly, luteolin treatment also arrested PC-3 cells in G(2)/M (p<0.01) and reduced the proportion of cells in G(0)/G(1) (p<0.05). This being the case, it is not surprising that this bioflavonoid also blocked the growth of subcutaneous PC-3 cell xenografts in athymic nude mice. These data demonstrate that naturally occurring type TI site antagonists such as luteolin are capable of inhibiting normal and malignant prostatic cell growth and proliferation in vitro and in viva and may possess prophylactic as well as therapeutic activities against prostatic proliferative diseases including benign prostatic hyperplasia (BPH) and prostate cancer.

Paclitaxel, a taxane, and vinorelbine, a semisynthetic vinca alkaloid drug, have tubulin as their common intracellular target but inhibit growth by acting with opposed mechanisms of action and binding to different sites. The purpose of this study was to evaluate in vitro the cytotoxicity of these two drugs as single agents, in combination and in sequence, against a human carcinoma ovarian cell line A2780S and its doxorubicin-resistant subline A2780R. The cell growth inhibitions were determined by the MTT assay. The cytotoxic effect of drug combinations at the IC50 level were analysed by the isobologram method of Steel and Peckham. On simultaneous exposure to paclitaxel and vinorelbine, synergistic effects were observed in A2780S and A2780R cell lines. On sequential exposure to paclitaxel first followed by vinorelbine or vice versa, a strong antagonistic interaction was observed. These data demonstrate that the interactions of vinorelbine and paclitaxel are highly schedule-dependent. These findings could have implications for the design of further clinical protocols and suggest that the simultaneous administration of the two agents may be the most suitable sequence while sequential administration may be avoided. Further preclinical and clinical studies are required to elucidate the relationship between vinorelbine and paclitaxel with regard to both anti-tumor activity and toxicity.

Hepatocyte growth factor (HGF) is a mesenchyme- or stroma-derived multipotent factor that regulates the growth, motility, and morphogenesis of various types of cells, including cancer cells. We investigated the effect of HGF on human breast cancer cells, and measured the concentration of HGF in the sera of breast cancer patients. When BT-20 cells were stimulated with HGF, the transmigration of cancer cells was markedly accelerated. In a checkboard assay, pronounced chemotaxic locomotion of BT-20 cells is expressed, corresponding to HGF concentrations. HGF treatment of BT-20 cells resulted in enhanced expression of alpha 2, alpha 3 and beta 1 integrin subunits, and augmented the binding activity to immobilized collagen. The c-met protein was expressed on the cancer cells in 48 of the 97 (49.5%) breast cancer primary tumors. In the serum, the advanced and recurrent cancer group showed a high level of this protein in comparison with the other patient groups. The mean value of serum HGF was 0.65 ng/ml in patients with distant metastases and 0.27 ng/ml in those with no such evidence. Thus, the HGF concentration becomes significantly elevated in the sera of patients with distant metastases. These findings suggest that HGF is involved in invasion and metastasis of breast cancer, and that serum HGF is useful as a tumor marker with a close correlation to the metastatic state of breast cancer.

Expression of E-cadherin, alpha-, beta- and gamma-catenins were studied in 100 patients with primary breast cancer compiled of 57 invasive ductal carcinomas (IDC) and 43 invasive lobular carcinomas (ILC) by means of immunohistochemistry. Loss of E-cadherin was observed in 26 (45.6%), and alpha-, beta- and gamma-catenin expression was lacking in 22 (38.6%), 27 (47.4%) and 22 (38.6%) IDCs, respectively. The expression in ILCs was significantly lower, as compared to IDCs (p<0.001). Immunostaining of both E-cadherin and catenins was completely lacking in 27 (47.4%) IDCs and 30 (93.8%) ILCs. Go-expression of E-cadherin/beta-catenin or E-cadherin/gamma-catenin was preserved more frequently than that of E-cadherin/alpha-catenin complexes. E-cadherin/catenin complex expression showed significant positive correlation with histological differentiation (p=0.037), ER (p=0.017) and PR expression (p=0.052), and negative correlation with c-erbB-2 receptor overexpression (p=0.046). Patients with tumours showing adhesion complexes containing alpha-catenin had an increased overall survival rate compared to other patients. Expression of either E-cadherin or alpha-catenin only, without the formation of entire adhesion complexes, was not correlated with overall survival. Thus, determination of both E-cadherin and catenins is suggested to add further information to estimate the prognosis of breast cancer patients.

Loss of the wild-type p53 activity and/or overexpression of the proto-oncogene bcl-2 are frequently detected in breast cancer and suggested to be related to chemotherapy and radiation therapy resistance. To identify the downstream signaling molecules for anti-proliferative and apoptotic activities of p53 and to investigate the interaction of bcl-2 with p53 in human breast epithelial cells, we have used the MCF10A cell line. We previously showed that overexpression of bcl-2 downregulates expression of p21(WAF1/CIP1) (a cyclin dependent kinase inhibitor which mediates p53 dependent G(1) arrest) and suppresses DNA damage-induced apoptosis in MCF10A cells. In the present study, we constitutively overexpressed p21(WAF1/CIP1) in bcl-2 overexpressing MCF10A cells to determine whether downregulation of p21(WAF1/CIP1) is necessary for the anti apoptotic activity of bcl-2, and to investigate the roles of p21(WAF1/CIP1) in p53-mediated cell death upon irradiation. Overexpression of p21(WAF1/CIP1) resulted in growth inhibition, but had no effect on bcl-2 inhibition of apoptosis following irradiation. Also, overexpression of p21(WAF1/CIP1) did not affect the dose-dependent radiation-induced cell lethality as determined by a clonogenic survival assay. These results suggest that bcl-2 downregulation of p21(WAF1/CIP1) is independent of the anti-apoptotic activity of bcl-2, and that p21(WAF/CIP1) is not involved in the p53-mediated cell death pathway.

Topoisomerase I inhibitors have shown positive effects in combination with radiation therapy in some studies. Normally oxygenated and hypoxic human MCF-7 breast carcinoma-cells were exposed to irinotecan (100 mu M or 250 mu M) or to SN-38 (10 mu M or 25 mu M) for 1 h prior to, during and for 3 h after radiation. Irinotecan and SN-38 showed little or no radiation sensitization of normally oxygenated MCF-7 cells but were effective radiation sensitizers of hypoxic cells. Both irinotecan and SN-38 diminished or eliminated the shoulder of the radiation survival curves of both the normally oxygenated and hypoxic cells indicating inhibition of the repair of sublethal radiation damage to DNA. Irinotecan (20 mg/kg or 30 mg/kg) was administered to mice bearing the EMT-6 mammary carcinoma on days 7 through 11 just prior to fractionated radiation (5x3 Gray). The tumor growth delays obtained with the combination regimens were greater than expected for simple additivity of the two treatments. Treatment with irinotecan resulted in decreased expression of topoisomerase I mRNA and increased expression of topoisomerase II mRNA in EMT-6 tumor tissue. Irinotecan treatment did not alter the protein levels for topoisomerase I or II in the tumor tissue; however, the combination of radiation therapy and irinotecan administration resulted in decreased topisomerase I and increased topoisomerase II protein in the tumor tissue. These results suggest that with appropriate scheduling of a topoisomerase I inhibitor and a topoisomerase II inhibitor with fractionated radiation therapy maximal cyto-reduction can be achieved.

Cigarette smoking plays a major role in the etiology of several human cancers. It is believed that formation of DNA adducts is an initial step in the carcinogenic process. In this study, we have examined the ability of dietary N-acetylcysteine (NAG) to inhibit the formation of cigarette smoke-related DNA adducts in various tissues of rats. Female Sprague-Dawley rats were exposed to cigarette smoke (10 mg TPM/m(3)) in a whole-body exposure chamber for 6 h per day, seven days a week for four weeks. The smoke-exposed groups were provided either an unrefined diet or diets supplemented with low (5,000 ppm) or high (20,000 ppm) dose of NAG. A sham group was given control diet and maintained on filtered ambient air. Tissue DNA analysis of smoke-exposed rats by nuclease P1-version of the P-32-postlabeling assay showed up to 6 adducts in the following descending order expressed as total adducts/10(10) nucleotides: 1 predominant (no. 5) and 4 (no. 1-no. 4) minor adducts in the (219 +/- 36), 6 minor adducts in the heart (93 +/- 11), 5 adducts in the trachea (50 +/- 16), and 4 adducts in the bladder (50 +/- 3.5); sham-treated animals showed 2 or 3 adducts in each tissue but at 4-20-fold lower levels. Dietary intervention with either high or low dose of NAC did not affect the levels of most adducts, except for the following: a 30-40% increase (P<0.05) for adducts 3 and 4 in the lung; a 40-50% decrease (P<0.05) for adduct 2 in the trachea; and a 30% increase (P<0.05) for adduct 2 in the bladder. In a second experiment conducted under identical conditions, most major and minor adducts remained unaffected with NAC intervention, except for adduct 2 in the trachea which was somewhat diminished. These results suggest that dietary NAC intervention does not significantly influence the levels of most major and minor adducts. However, some minor adducts in the lung, trachea and bladder were modulated differentially.

In order to determine the physiological significance of c-mos RNA expression in somatic cells, we introduced antisense c-mos under the control of an inducible promoter. NIH/3T3 cells were stably transfected with antisense mos under the control of the mouse mammary tumor virus long terminal repeat (MMTV-LTR). Positive transfectants were selected under G418 conditions. Following selection, NIH/3T3 cells that received the antisense mos failed to form foci, whereas sense mos transfected cells grew normally. Moreover, v-mos-transformed cells were unaffected by antisense mos transfection. Of: interest, NIH/3T3 antisense mos transfectants that survived selection were growth-arrested. Nuclear abnormalities and the extrusion of microvesicles containing cellular material were observed in these cells. In order to rescue these cells from growth inhibition, the v-mos gene was introduced into cells by acute infection with Moloney murine sarcoma virus. Following infection, these cells resumed growth and became rapidly transformed. In other experiments, mouse C2 cells stably transfected with antisense mos showed a slower growth rate and gross morphological changes. C2 cells containing antisense mos under the control of mouse metallothionein-1 promoter had a large and flattened morphology and a relatively high percentage (30%) of binucleated cells. Our results indicate that basal level expression of antisense mos (under uninduced conditions) results in either arrested or retarded cell growth. The phenotypes exhibited in both cell lines leads us to suggest that the c-mos expression may play a role in mitotic progression in some somatic cells, in particular affecting cytokinesis.

Retinoids have antiproliferative effects on epithelial cells and have been used as chemopreventive and chemotherapeutic agents for several human cancers. Retinoid/interferon combinations have demonstrated activity in advanced stage cervical cancer. The objective of this study was to quantify and localize the expression of RAR-beta 2, a retinoid inducible receptor, in normal cervix and cervical squamous cell carcinoma by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and in situ RT-PCR. Specimens where obtained from 11 patients enrolled in a clinical trial to test all-trans retinoic acid (tRA) in combination with interferon-alpha 2a (IFN-alpha 2a) in the treatment of metastatic or recurrent cervical carcinoma. Expression of RAR-beta 2 in cervical carcinoma and normal cervix was measured by quantitative RT-PCR. DNA competitors were used to estimate the relative expression level of RAR-beta 2. Expression of RAR-beta 2 was examined in normal cervix by in situ RT-PCR. Expression of RAR-beta 2 in cervical carcinoma ranged from 0.33 to 1.40 with a mean of 0.89+/-0.13 vs. 1.0+/-0.13 for normal cervix (NS) with RAR-beta 2 reduced to less than or equal to 65% in five cases. Irt situ RT-PCR identified RAR-beta 2 most prominently in basal and para-basal epithelial cell layers of normal exocervix; stromal expression was markedly decreased. This is the first report to localize expression of RAR-beta 2 mRNA in normal cervical epithelium and quantify expression in normal cervix and cervical squamous cell carcinoma. Because retinoid receptors are the proximate mediators of retinoid action on gene expression, alteration of their expression or function could result in cancer development.

We examined effects of ibuprofen on the growth and development of DMBA-induced mammary cancers in mature female Sprague-Dawley rats. Ibuprofen was added to the standard diet at approximately 1,000 mg/kg rodent chow, resulting in an average daily dose of 25 mg per day per 0.25 kg rat. After five weeks of ibuprofen treatment, there was a 37% reduction in tumor volume compared to a 260% increase in the volume of tumors in untreated rats (p<0.001). These results suggest that ibuprofen may have potential in the chemoprevention and treatment of breast cancer.

Antibodies to human T lymphotropic virus type I has been detected in subjects of different human ethnicities all around the world. Etiological relationship between the virus and human diseases has been claimed by many investigators. Amplified and sequenced region of the long terminal repeat of human T lymphotropic virus type I obtained from nucleic acid extracted from serum samples of Japanese and Brazilian patients with cancer of uterine cervix and normal Brazilian subjects, all seropositives for the virus, showed minor genetic variations when compared to the Japanese prototype.

Cathepsin B (CB), a lysosomal cysteine proteinase, is implicated in tumour invasion and metastasis. Although direct exposure of THP-1 cells to arachidonic acid did not stimulate the induction of CB activity, cells treated with arachidonic acid followed by interferon-gamma (IFN-gamma), as well as concurrently treated cultures with arachidonic acid and lipopolysaccharide (LPS) or phorbol ester (PMA) increased CB activity in a dose-dependent manner. LPS and IFN-gamma-induced increases in CB were down-regulated by dexamethasone, an inhibitor of phospholipase A(2) (PLA(2)). Whereas dibutyryl cAMP, which increases PLA(2) activity, caused elevations in CB in THP-1 cells; inhibition of protein kinase A by H-89, which reduces PLA(2) expression, blocked the effect of dibutyryl cAMP. On the other hand, indomethacin, an inhibitor of cyclooxygenase, and ketoconazole, an inhibitor of lipoxygenase, up-regulated CB activity dose-dependently, indicating that the balance among PLA(2), cyclooxygenase and lipoxygenase activities might regulate the levels of CB synthesis. These data suggest that arachidonic acid may be associated with part of the intracellular signal pathway in the induction of CB activity by LPS, PMA and IFN-gamma.

We examined argyrophil nuclear organizer regions (AgNOR) and proliferating cell nuclear antigen (PCNA) in 41 patients with surgically-treated thymic disease. AgNOR count and PCNA labeling index (LI) in thymic carcinoma were significantly higher than those in thymoma and thymic hyperplasia. A positive correlation was observed between the PCNA LI and argyrophilic nucleolar organizer region (AgNOR) counts within all thymic disease (r=0.31, P=0.002). The PCNA LI in invasive thymoma was lower than that in non-invasive thymoma. In survival analysis, the cut-off values for the PCNA LI and AgNOR count were chosen to produce two categories with equal numbers of 26 thymoma patients. There were no significant difference in the survival rate between the lower and higher group patients in relation with AgNOR count and PCNA LI. We conclude that combining AgNOR and PCNA may discriminate the biological activity of thymic disease. These staining methods can be performed with ease and, applied in a clinical laboratory on a routine basis to help predict cytological malignancy of thymic disease.

c-erbB was introduced into normal human fibroblasts, MRC-5, which expressed normal levels of EGF receptor and in a SV40-transformed cell line, MRC-5V1, derived from them, which expressed markedly reduced levels of EGF receptor mRNA. MRC-5 overexpressing c-erbB, responded mitogenically to EGF. However, addition of high EGF concentrations markedly reduced DNA synthesis and resulted in the inhibition of cellular growth. In contrast, MRC-5V1 exhibited an increase in DNA synthesis in an EGF-dependent manner which was enhanced by overexpression of c-erbB. These cells, unlike MRC-5, also produced TGF alpha, an EGF receptor ligand which is often associated with cellular transformation. Ligand-activation of EGF receptor did not alter the lifespan, induce focus formation or anchorage-independence of MRC-5 and all the cell types remained non-tumourigenic in nude mice. However, c-erbB induced the expression of tPA, c-jun and junB in both MRC-5 and MRC-5V1. The data suggest that overexpression and activation of c-erbB is unlikely to play a role in immortalisation of human diploid fibroblasts but it may contribute to cellular transformation.

Chemotherapy has proven to be effective in the management of high grade non-Hodgkin's lymphoma (NHL). However drug resistance remains a major clinical obstacle to effective disease control. One such well documented system involves multidrug resistance (MDR), characterized by the overexpression of a 170 kDa membrane protein termed P-glycoprotein (Pgp). Analysis of P-glycoprotein was done on lymph node biopsies obtained from 109 patients with high grade NHL. Patients were followed up for time periods ranging from 7 to 26 months after chemotherapy or until recurrent/residual disease was diagnosed. Analysis of Pgp in tissue samples was carried out by immunocytochemistry and Western blot. Of the 109 patients, 36 had either residual or recurrent disease. All these patients had a second lymph node biopsy done which was also analyzed for Pgp. P-glycoprotein was detected by immunocytochemistry in only 5 of the 73 patients who remained disease-free, while it was expressed in 26 of the 36 patients with residual/recurrent disease. All the 36 tissue samples of the latter group and 42 of the 73 biopsies of the disease-free group were re-analyzed by Western blot. Only one of the 42 samples from the disease-free group showed a positive Western blot reaction while 30 of the 36 samples from patients with recurrent/residual disease gave a positive reaction. Thirty-four of the 36 repeat biopsy samples from patients with recurrent/residual disease were positive for Pgp. Correlation analysis, thus showed significant relationship between prognosis and detection of Pgp by immunocytochemistry (r=0.85, p<0.001) and Western blot (r=0.90, p<0.001). Moreover, the odds ratio of a tumor positive for Pgp not responding to chemotherapy was 35.36 (CI 11.03, 113.37). Thus evaluation for Pgp may prove to be a useful prognostic marker for high grade NHL, and may also prove useful in designing or altering chemotherapy protocols in such patients.

Possible pathways of the phosphorylation of 5-fluorouracil (FUra) were investigated in vitro and in vivo, using certain xenografts, of human gastric and colorectal cancer cell lines. The oxonic acid (Oxo), an inhibitor of orotate phosphoribosyltransferase (OPRTase) that catalyzes the direct production of 5-fluorouridine 5'-monophosphate from FUra (the first pathway) and 2, 6-dihydroxypyridine (DP), an inhibitor of uridine phosphorylase (UPase), the enzyme converting FUra to 5-fluorouridine (the second pathway), were employed to estimate a contribution of these two metabolic pathways in the anabolism of FUra. Ten out of 13 cancer cell lines tested were found to utilize the first route for the phosphorylation of FUra, as revealed by marked inhibition of the phosphorylation of FUra by Oxo in 4 of 5 and in 6 of 8 gastric and colorectal cancer cell lines, respectively. The phosphorylation of FUra in the xenografts of human AZ521 gastric adenocarcinoma and SNU-C2A colorectal carcinoma was also regulated by Oxo, the production of 5-fluoro-nucleotides after i.v. injection of FUra with the Oxo significantly decreased from 0.587 to 0.311 nmol/g and from 1.75 to 0.40 nmol/g in respective xenografts, suggesting that the nature of an anabolic pathway of FUra in the tumor cells in vitro reflects the metabolic pattern found in the in vivo conditions. Moreover, the intracellular concentrations of phosphoribosylpyrophosphate (PPRibP) in DLD-1 and SNU-C(2)A cells were much higher than those found in HCT-15 and MKN-28 cells, leading to the findings that FUra was phosphorylated by OPRTase in the former and by UPase and uridine kinase in the latter cells. These results also may suggest that the intracellular levels of PPRibP in the tumor cells are importantly related to the selection of a proper metabolic pathway of FUra by the cell.

Integrin alpha 6 is believed to be involved in malignant biological development. High tumor/normal (T/N) ratio of integrin alpha 6 was observed in intestinal type tumors in gastric carcinoma. In an immunohistochemical study, an intense and continuous staining was seen at the basement layer in the intestinal type tumors. Concerning the variant, there was a significant correlation between the expression ratio of variant B/variant A and the depth of tumor invasion. In conclusion, i) integrin alpha 6 expression was intimately correlated with histologic differentiation, and ii) the overexpression of variant B may be correlated with tumor invasion of gastric carcinoma.

Chest wall resection (CWR) is occasionally required in cancer patients and may cause significant functional deficits. We report an instance where a rigid orthotic device was useful in rehabilitation. Such custom-made devices can improve the quality of life for cancer patients.

Breast cancer is the most common malignancy seen in women. Due to the increasing awareness for this disease and the increasing use of screening mammography, more patients are now diagnosed with early stage disease. Over the past few years, the indications for adjuvant treatment have expanded considerably, and more women are now candidates for chemotherapy, hormonal therapy, or both. The role of various prognostic factors and their implications on the design of a multidisciplinary therapeutic approach is discussed in this review. Current recommendations regarding the extent of the surgery, the role of radiation, and its integration to the adjuvant treatment are discussed. Guidelines on adjuvant chemotherapy and adjuvant hormonal therapy are also being presented.

The uniqueness of the complementarity determining region III (CDRIII) has been utilized successfully in the last decade for development of a patient specific, molecular, polymerase chain reaction (PCR)-based assay for determining minimal residual disease in various lymphoid malignancies. There are various approaches for carrying out this test. i) CDRIII primers are used to amplify the corresponding DNA from the same patient and quantitation of the amplified CDRIII bands is done by generation a standard curve of known amounts of purified patient's tumor DNA, followed by a linear regression analysis to quantitate the results. ii) CDRIII primers are used to amplify a serially-diluted patient's sample (unknown), with replicate points. According to Poisson equation, replicate points in each dilution can be either all positive, all negative, or 'mixed', negative and positive. The quantitation, according to this approach is done by determination of the dilution point where there are 'mixed' lanes plus the flanking 'all negative' and 'all positive' lanes, assuming that the test can always detect one tumor cell in 100,000 cells. In this communication we show evidence that the use of the Poisson method can lead to an underestimation of the amount of tumor cells, due to the great variability in the priming and amplification among the various CDRIII primers. This variation is inherent to the size, C/G ratio, melting point of each primer, etc. In a simulated statistical model we show that the magnitude of error in the Poisson method could reach 1-2 logs. In contrast, using the standard curve for each patient and regression analysis eliminate these problems.

The prognostic significance of pack-years smoked (PY) and family history of cancer (FH) was studied using the Cox proportional hazard model in 970 patients with non-small cell lung cancer. PY influenced survival only in women after other variables were adjusted [Hazard ratio (HR): 1.75 (1.45-2.11)]. FH also influenced survival with marginal significance in women with stage I disease [HR: 2.17 (0.89-5.29), p=0.088]. Furthermore, these two factors operated additively in women [HR for patients with both smoking and family histories over those without either: 2.25 (1.18-4.26)], but not in men.

The p53 protein is a transcription factor that is frequently mutated in human malignancies. Using the MCF10AT model for early human breast cancer we show that P53 protein is unmutated indicating that mutations are not necessary for alterations in growth and morphology that accompany preneoplastic stages of breast tumor progression. Although p53 protein is wild-type in cells of the MCF10AT model system, it exists predominantly in a conformationally altered state that is defective in its ability both to bind DNA in a sequence-specific manner and to induce transcriptional activation from the WAF-1 promoter. This contrasts with P53 from the non-tumorigenic parental MCF10A cells which is predominantly conformationally normal and functionally active. The possibility that stabilized wild-type but conformationally altered P53 plays a role in the neoplastic progression of preneoplastic MCF10AT system cells is discussed.

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.

The fraction of glucose metabolized to lactate is dramatically reduced during erythroid differentiation of mouse erythroleukemia (MEL) cells induced by dimethyl sulfoxide (DMSO), hexamethylene bisacetamide (HMBA), or sodium butyrate treatment. In order to determine the mechanism of the reduction in lactate production, several enzymatic steps in glucose catabolism were investigated. No changes in glycolytic enzyme levels were found during differentiation that could account for the alteration in lactate production and alterations in pyruvate kinase activity are known not to occur during MEL cell differentiation. Further, utilizing D-mannoheptulose, a specific inhibitor of hepatic-/tumor-specific glucokinase, no dependence on the activity of this enzyme for growth or differentiation was observed. Therefore, the possibility was entertained that the decrease in lactate production reflected a decrease in fructose 2,6-bisphosphate (F-2,6-P-2) which is a major regulator of the lactate production due to its ability to allosterically stimulate phosphofructokinase-1 (PFK-1) activity. PFK-1 cannot function in the absence of F-2,6-P-2 when only a suboptimal concentration of one of its substrates, fructose-6-phosphate (F-6-P), is present. When assayed under limiting F-6-P concentrations, it was found that following DMSO- or HMBA-induced differentiation, PFK-1 activity was decreased 7-20-fold. This finding suggested that F-2,6-P-2 levels might be controlling lactate production in this system. In keeping with this idea, marked decreases in F-2,6-P-2 levels were found to occur during DMSO- or HMBA-induced differentiation. These data suggest that decreasing F-2,6-P-2 levels account for the decrease in lactate accumulation that occurs during MEL cell differentiation.

The initiation and metastatic progression of human cancers are genetically controlled. We therefore sought to identify specific chromosomal changes associated with the development of a melanoma and its metastasis to the bladder in a 57-year-old Caucasian man by analyzing his lymphocytes and metastatic melanoma cells. Approximately 4% of the patient's PHA-stimulated lymphocytes showed constitutional abnormalities of chromosomes 1, 2, 3, 4, 7, 9, 10, 12, 14 and 19. On the other hand, his melanoma cells showed clonal markers involving chromosomes 2, 4, 5, 6, 7, 9, 10, 14, 16, 17 and 20 plus many more. Our results indicate that both primary chromosome abnormalities and some of the secondary cytogenetic defects of melanoma cells can be identified in lymphocyte cultures and that such abnormalities may be possible markers for early diagnosis and treatment.

Selection of cells for resistance to cisplatin results in resistance to arsenite and selenite. Mammalian cells detoxify arsenite and selenite by S-adenosylmethionine dependent methylation. We aimed to investigate whether S-adenosylmethionine dependent methylation is involved in the cellular metabolism of cisplatin. Treatment of human ovarian cancer cells 2008 and the cisplatin-resistant subline 2008/C13*5.25 with the S-adenosylhomocysteine hydrolase inhibitor adenosine-dialdehyde, an indirect inhibitor of transmethylation, resulted in a significant elevation (16-fold in 2008, 6-fold in 2008/C13*5.25) in the cellular content of S-adenosylhomocysteine without changing S-adenosylmethionine. Adenosine dialdehyde synergistically enhanced the cytotoxicity of cisplatin and carboplatin as evidenced by combination indices <1 using the combination index-isobologram method in clonogenic assays with 2008 human ovarian adenocarcinoma cells. However, the cellular accumulation, efflux, steady state content, and the formation of DNA adducts of the cisplatin [H-3]-DEP were not affected by adenosine-dialdehyde. Sodium arsenite was significantly more toxic in mice pretreated with adenosine-dialdehyde, whereas the toxicity of cisplatin remained unchanged. These studies suggest that inhibition of S-adenosylmethionine dependent transmethylation enhanced the toxicity of cisplatin and carboplatin to human ovarian carcinoma cells in vitro without directly affecting the metabolism of either platinum drug.

Cell cycle regulators have recently been implicated in oncogenic transformation of cells, including the cyclins active in the G1 phase of the cell cycle and their respective cyclin-dependent kinases (CDK) whose activities are regulated by a set of inhibitors of CDK (CDKI). Since CDKIs can inhibit cell proliferation, they may have a role as tumor suppressor genes. To determine if alterations of CDKI genes may be involved in tumorigenesis of breast cancer, we examined the mutational status of p16(INK4A), p15(INK4B), p18(INK4C), p19(INK4D) CDKI genes in 36 primary breast carcinomas and 9 breast cancer cell lines using polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP), direct DNA sequencing, and Southern blot analysis. Furthermore, amplification of cyclin D1, D2, D3 genes were also examined in these samples. One mutation of p15(INK4B) gene occurred, resulting in change of aspartic acid to asparagine at codon 85. Since aspartic acid at this position is conserved between all four human and murine INK4 proteins, this missense mutation may have functional significance. The sample with a p15(INK4B) point mutation was accompanied by amplification of the cyclin D1 gene. A deletion of the p18(INK4C) gene was found in a primary tumor. Three deletions of the p16(INK4A) gene and two deletions of the p15(INK4B) gene were found in the cell lines. Also, we found amplification of the p15(INK4B) and p16(INK4A) loci in a clinical sample as well as amplification of the p19(INK4D) in another sample, and amplification of the myeloperoxidase (MPO) gene in one cell line and two primary tumors. We suspect that a critical gene for breast cancer is amplified near the MPO gene. These data indicate that CDKI mutations are moderately rare in breast cancer and are often associated with the simultaneous alteration of more than one cell-cycle regulatory gene.

The double-stranded RNA dependent eIF-2 alpha kinase (PKR) has been implicated in the regulation of a number of cellular processes including cell growth and differentiation. Previous studies using embryonic mouse 3T3-F442A cells have indicated that PKR undergoes phosphorylation and activation in vivo. This activation of PKR has been attributed to a subset of poly(A)(+)-rich cellular RNA (R-RNA) having sufficient secondary structure to interact with the kinase. To characterize the R-RNA activity, a cDNA was prepared which, when transcribed in vitro, gave rise to an RNA transcript that retained its property to activate PKR. The ability of the transcript to activate PKR was sensitive to ribonuclease V1 and was abolished by the addition of high concentrations of poly(I).poly(C). The cloned cDNA was utilized for liquid RNA/DNA hybridization experiments, which disrupted the secondary structure of the R-RNA and for Northern blot analysis. The results from these studies indicated that the measurable RRNA activity was represented in a specific cellular RNA which was responsible for the activation of PKR. Furthermore it was found that the R-RNA was specifically associated with PKR and that this complex could be detected directly in cell extracts. The nucleotide sequence of the cDNA was determined. We propose that this novel cellular RNA may play a critical role in regulating the activation of PKR and thus be an important component in the control of cell growth and differentiation.

p21(WAF1/CIP1) is a potent inhibitor of various cyclin-dependent kinases, the expression of which is transcriptionally regulated by tumor suppressor gene product p53. We immunohistochemically examined the expression of p53 and p21(WAF1/CIP1) in 61 esophageal squamous cell carcinomas. p53 protein was expressed in 37 (61%) of 61 carcinomas. p21(WAF1/CIP1) was consistently expressed in the normal stratified esophageal mucosa. In the carcinomas, the expression of p21(WAF1/CIP1) protein was markedly reduced or not expressed in 33 (54%) cases. Clinicopathologic analyses revealed that no significant correlation exists either between p53-positive and -negative cases or between p21(WAF1)/(CIP1)-positive and -negative cases. Twenty-four cases were p53-positive/p21(WAF1/CIP1) negative, 15 were p53-negative/p21(WAF1/CIP1)-positive, 13 were positive for both and 9 were negative for both, and these findings thus showed an inverse correlation of the positivity between p53 and p21(WAF1/CIP1) (p<0.05). Furthermore, of the 13 cases with positive staining for both, the distribution of the expression was mutually exclusive in 6 cases and coincidental in the remaining 7 cases. These findings showed the p53-dependent expression of p21(WAF1/CIP1) was observed in esophageal squamous cell carcinomas, while the lack of an absolute correlation between abnormal p53 expression and p21(WAF1/CIP1) expression suggested that the p53-independent expression of p21(WAF1/CIP1) might also occur in some portions of the esophageal squamous carcinomas, suggesting that the molecular mechanisms of esophageal carcinogenesis appear to be complicated.

We compared the DNA content in both the primary and recurrent lesions of 12 cases with colorectal cancer. The DNA ploidy agreed in 10 cases (83.3%) between the primary and recurrent lesions. The mean DNA index was similar between both lesions. The DNA content seems to remain stable during recurrence in many cases of colorectal cancer. However, the existence of variations in the content also suggests that the specimens should be investigated for both primary and recurrent lesions in order to obtain more information on the biological or clinical behavior of the tumor.

In vitro chemosensitivity testing using a collagen gel droplet embedded culture drug sensitivity test (CD-DST), was conducted with several types of solid cancer. The overall evaluable rate was 80% (443/554), including 76% for lung (n=243), 78% for breast (n=110), 87% for gastric (n=62), 83% for colorectal (n=107) cancers and 88% for 32 metastatic brain tumors. The in vitro sensitivity of breast, gastric and colorectal cancers to mitomycin C (MMC), cisplatin (CDDP), 5-fluorouracil (5-FU) and doxorubicin (DXR) was similar to the efficacy rates reported for each drug. This was also observed with lung cancer, the sensitivity of which to MMC, CDDP, vindesine (VDS) and etoposide (VP-16) was similar to the clinical efficacy. The clinical response to chemotherapy was compared with the results of in vitro chemosensitivity testing in Il patients: the clinical correlation was 91%, with a 80% true positive and 100% true negative rate. These results suggest that the CD-DST may be clinically useful by allowing the prediction of clinical response in various solid cancers.

We conducted an incremental cost-effectiveness analysis to evaluate an adjuvant multimodal therapy (chemotherapy + radiotherapy) in patients with esophageal adenocarcinoma undergoing surgery. We utilized the clinical data of a published controlled trial comparing preoperative chemotherapy + radiotherapy versus surgery alone. Information on costs was derived from local data and verified against reported values. Multimodal treatment was found to improve life expectancy by 196.9 discounted years every 100 subjects (survival gain of about 2 years per patient). Costs of this neoadjuvant therapy were estimated as $780,010 per 100 patients. Our cost-effectiveness analysis showed that the cost per life year gained was $3,961 for the multimodal therapy in comparison with surgery alone. The pharmacoeconomic profile of this therapeutic modality compares favorably with previous economic data calculated for other types of health care intervention.

The tumor metastatic ability and tumorigenicity of the mouse fibrosarcoma cell line (FSa-II) were significantly reduced due to overexpression of manganese superoxide dismutase (MnSOD) as reported previously. We investigated changes in the in vitro basic character of FSa-II cells transfected with the human MnSOD cDNA which was employed in the previous studies. FSa-II and the control vector-transfected cell line NEO, had no detectable MnSOD activity. SOD-H, into which MnSOD cDNA was transfected, is the cell line with high MnSOD activity. The malignant phenotype, characterized by serum-independence, was suppressed with elevated MnSOD activity. A quantitative comparison of transferrin receptor (TfR) by flow cytometry showed the amount of TfR on the membrane of SOD-H cells to be significantly less than that on the membrane of NEO cells. The amount of CD44 expression on SOD-H cells was almost the same as that on NEO cells. The results of this study suggest that the overexpression of MnSOD is related to suppression of the malignant phenotype and that changes of iron metabolism may play an important role in this process.

We recently reported that three members of the integrin family of cell adhesion molecules, designated alpha 2 beta 1, alpha 3 beta 1, and alpha 6 beta 4, are expressed at increased levels within the tumors and cell lines of patients with SCC. These three integrins have been reported to serve as receptors for laminin isoforms, and we also previously observed that laminins are secreted by SCC cell lines isolated from patients. In this study, the expression and localization of the three integrins and laminin in situ was evaluated in ten tumor specimens from patients with SCC by immunohistochemistry using integrin subunit-specific monoclonal antibodies. The ability of the antibodies to inhibit laminin attachment of a human squamous cell carcinoma line was determined by in vitro cell adhesion assay. Laminin and the three integrins were co-localized along the invasive border of the tumor parenchyma in 10/10 patient tumor specimens. Attachment of the UM-SCC-38 cell line to laminin was strongly inhibited by specific mAbs to alpha 2 and alpha 6 integrin subunits alone, or completely using a combination of alpha 2, alpha 3, and alpha 6 subunit specific mAbs. The co-localization of the three abnormally expressed integrins and laminin in patient tumor specimens indicates the potential for interaction of these receptors and ligand in vivo. The results of the cell adhesion assays using a patient SCC cell line that expresses the same repertoire of integrins confirms that SCC attach to laminin isoforms primarily through the alpha 2, alpha 3 and alpha 6 subunit-containing integrins. These findings provide a basis for undertaking experimental studies to obtain small molecule receptor antagonists to determine the role of these integrins in tumor formation, growth, invasion and metastasis in vivo.

To clarify the time trends in the characteristics of colorectal cancer, we investigated 1,286 Japanese patients with colorectal cancer who underwent a resection of the disease between 1972 and 1995 at our hospital. Based on our findings, this carcinoma tended to shift to the more proximal colon in the second 11-year period in comparison to the first 11-year period (P=0.0001). This tendency was also found in elderly patients (P=0.0001), but not in young ones. These findings thus suggest the need for targeted examinations for the early detection of the proximal colon cancer, especially in the elderly.

The hMSH2 gene participates in DNA mismatch repair and its mutation can result in genetic instability of the human genome which is an important feature of tumorigenesis. In this study, genetic alterations of the hMSH2 gene were examined in 43 ovarian, 36 non-small cell lung (NSCL), 31 poorly differentiated gastric, 15 endometrial, and 11 colon cancers, nine gastric cancer cell lines, 41 adult T-cell leukemias (ATLs), two ATL cell lines, and 37 non-Hodgkin's lymphomas (NHLs), using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) technique. Microsatellite instability (MSI) was also investigated for ovarian, NSCL, and colon cancers. The incidence of MSI was 1/36 (3%) for NSCL, 2/23 (9%) for ovarian, and 1/11 (9%) for colon cancers. Missense base changes of the hMSH2 gene were identified in two gastric cancer patients (ATG to ATA resulting in Met changing to Ile at codon 688 in exon 13 and ACA to GCA resulting in Thr changing to Ala at codon 803 in exon 14). These mutations were found in samples with no MSI. One ovarian and one gastric cancer, and six ATL samples showed two types of polymorphisms of hMSH2 (CTT to TTT resulting in Leu changing to Phe at codon 390 in exon 7 and CAG to AAG resulting in Gin to Arg at codon 419 in exon 7). Our data suggest that MSI and hMSH2 mutations are uncommon in sporadic tumors.

Acute lymphoblastic leukemia (ALL) is the most frequent childhood tumor. Although the cure rate has increased dramatically over the past few years, the molecular and cellular biology of this disease is still not fully understood. Important factors affecting prognosis include age, leucocyte count, cytogenetic abnormalities, immunophenotype and sex. However, the role of cell kinetics, tumor associated genes and drug resistance have rarely been investigated in pediatric ALL. This preliminary study looks at these aspects in this disease. Immunocytochemical analysis of the tumor suppressor protein p53, the anti apoptotic protein bcl-2, the multidrug resistance (MDR) gene product P-glycoprotein and the proliferation marker, Ki-67 were done using appropriate monoclonal antibodies. A total of 88 previously untreated children with confirmed ALL diagnosis and 40 age matched controls were included in the study. Ki-67 was expressed in 81% and P-glycoprotein in 68% of all cases. P53 expression was observed in 68% and Bcl-2 in 50% of cases. Fifty percent of all cases also showed concomitant expression of bcl-2 and p53. Since wild-type p53 is known to negatively regulate the expression of bcl-2, the co-expression of these two proteins having contrasting functions suggests that the p53 may be of a mutant variety. In addition, the expression of p53 also correlates to the presence of the multidrug gene product P-glycoprotein. Control samples were negative for all the four markers. P53, bcl-2 and P-glycoprotein can all influence chemotherapy resistance, the first two by regulating programmed cell death and the third by influencing intracellular drug levels. All patients are now being followed up to assess treatment response and to study the prognostic significance of the analyzed markers.

We have recently reported increased concentrations of polychlorinated biphenyls and chlordane compounds in adipose tissue from patients with non-Hodgkin's lymphoma (NHL) compared with surgical controls. As a feasibility project we have now studied the occurrence of polybrominated diphenyls ethers (PBDE) in the same study population. Four samples of adipose tissue were selected at random from the case and control series. We were able to identify PBDEs in all samples. PBDEs are environmental contaminants and the levels are increasing in sediment and biological samples. Our results indicate that human beings are also exposed, mainly through the food chain, resulting in ng/g concentrations of PBDEs in the adipose tissue. This has not been previously reported from Sweden. Any health consequences are so far not studied. The environmental situation for PBDEs is thus similar to that of PCBs a few decades ago.

To investigate radiosensitization by halogenated pyrimidines in human cells, SW-1573 lung tumor cells were grown in absence or presence of 4 mu M of iododeoxyuridine (IdUrd). Cell survival after irradiation with gamma-rays from a Cs-137 source with single doses up to 8 Gy, was determined by clonogenic assay, Radiosensitization by IdUrd was observed in both exponentially growing and plateau phase cells. Linear-quadratic analyses of the radiation survival curves demonstrated that radiosensitization was mainly expressed by an increase of the value of alpha (determining the initial slope of the survival curve) which can be interpreted as an enhancement of fixation of potentially lethal damage (PLD). The value of beta (determining the curvature of the survival curve), which might be attributed to enhanced interaction or to fixation of sublethal damage (SLD), was not influenced. In view of clinical applications of IdUrd it is of interest that radiosensitization has its main effect in the low dose region of the cell survival curve, described by the linear term alpha D.

Epidermal growth factor (EGF) is an important growth factor regulating both normal and malignant cells. The present study analyzes the expression pattern of EGF and its receptor (EGF-R) in 424 cases of various stages of tumor progression in the uterine cervix. In all the samples studied, EGF and EGF-R expression was predominant in the basal or basaloid cells. Increased expression of the two proteins correlated with increasing histological abnormality (dysplasia). In invasive cancer, alteration of EGF expression was more evident than EGF-R. The expression pattern showed significant correlation in the basal and spinal cell layers. This increased expression of EGF observed in dysplastic and malignant cells may be due to overexpression of EGF-R and suggests its role in cellular proliferation.

Albumin dominates the nitrogen and energy resources in blood. However, only limited data is available on its accumulation and catabolism by tumors. This was caused by the lack of suitable radiolabels for long-term follow-up of protein catabolism in vivo. Conventional radiolabels like radioiodine are metabolically unstable. After lysosomal degradation diffusible tracer residues are rapidly released from catabolic sites, Tumors with high metabolic activity evade detection. To study the uptake of rat serum albumin (RSA) by tumors a conventional radioiodine label and two residualizing radiolabels were chosen. It is known that residualizing I-131-tyramine-deoxisorbitol and In-111-DTPA protein labels remain trapped at catabolic sites after lysosomal degradation of their carrier proteins. We were able to show by scintigraphy and after organ removal that a Walker-256 carcinosarcoma with a turner size of about 5% of the body weight accumulated more than 20% of the initially injected dose of a In-111-DTPA-RSA within 24 h. Tumor uptake rates for albumin exceeded those of the kidneys by about 4 times, and those of the liver by about 3 times. It was estimated that about one out of two albumin molecules trapped by an ovarian-342 tumor must have been degraded during 72 h. High uptake and degradation rates would make albumin an alternative nitrogen and energy source for these tumors. Although an unfavorable time-frame limits the use of residualizingly labeled albumin for scintigraphic tumor diagnosis in man, albumin might be an interesting carrier for delivering covalently attached chemotherapeutic agents into tumors by an alternative lysosomal route.

Limb sparing surgery has replaced amputation surgery for treating sarcomas of the lower limb in most cases. Wide resection followed by postoperative radiation therapy can achieve acceptable local control and survival rates in patients with bone and soft-tissue sarcomas of the lower limb. Recurrent or persistent disease constitutes a major oncological problem. Local symptoms such as agonizing pain, fractures, tumor fungation, inability to walk and inability to maintain daily activities, further impair the patient's quality of life. In this clinical set-up palliative amputation of the limb should be considered. Fourteen patients with soft-tissue or bone sarcomas underwent palliative major amputation. The procedures included: hemipelvectomy, hip disarticulation, knee disarticulation, above or below-knee amputation. Local control of the disease and pain, and improvement of the performance status were observed in 13 evaluable patients. The mobility was restored in 13/14 patients. The median survival following the procedure was 9 months. There was only one case of immediate post-operative death. Severe phantom pain was not reported by any of the patients. Quality of life was reported to be improved by two-thirds of the patients. We found palliative major amputation surgery worth-performing in low-performance status cancer patients with locally advanced disease of the lower limb.

Loss of DNA mismatch repair is a common finding in hereditary nonpolyposis colon cancer as well as in many types of sporadic human tumors. The effect of loss of DNA mismatch repair activity on sensitivity to cisplatin and carboplatin was tested using MSH2 and PMS2 knockout cell lines. The knockout dMsh2 embryonic stem cell line was 2.1-fold more resistant to cisplatin and 1.7-fold more resistant to carboplatin when compared to the isogenic wild-type wt-2 cell line. Likewise, the PMS2(-/-) mouse fibroblasts were 1.9-fold more resistant to cisplatin and 1.5-fold more resistant to carboplatin when compared to the isogenic PMS2(+/+) fibroblasts. These findings demonstrate that loss of mismatch repair due to knockout of either MSH2 or PMS2 results in low-level resistance to cisplatin and carboplatin, drugs that form the same types of adducts in DNA. These data validate results previously obtained using non-isogenic mismatch repair-proficient and -deficient cell lines, and indicate that simple recognition of the cisplatin adduct by the MSH2/MSH6 heterodimer is not sufficient for full detector function, but that PMS2 is also required for the pro-apoptotic signal to be generated from this detector.

The aim of the present study was to develop an animal model to test the therapeutic potential of purified CD4 and CD8 T-lymphocytes against the intracerebrally implanted rat glioma cell line TZ363. Peripheral immunization of donor rats was performed by subcutaneous injection of viable TZ363 tumor cells while control animals received buffer injection. Donor splenic T-lymphocytes were prepared 14 days later and enriched by immune-bead MACS sorting. FACScan analysis revealed that of the pooled and sorted cells 91% of the tumor immune group were T-lympocytes and from the control animals 96%. The purified immune CD4/CD8 T-lymphocytes (1.2 to 5x10(7) cells) were injected intraperitoneally into 12 adult rats (three groups; each four animals), which were challenged five days later by an intracerebral injection of 5x10(4) TZ363 glioma cells. Four rats received 1.4x10(7) T-cells from control animals. While 3 of 4 animals developed a brain tumor and died in the control group, all animals, which received 5x10(7) immune T-cells survived the intracerebral tumor challenge. In the other groups survival rate depended on the amount of T-cells given. All other rats were sacrificed 32 days after intracerebral grafting. No tumor was found in these animals. Our data demonstrate that an anti-tumor T-cell response can be raised against the malignant rat glioma TZ363 and that purified CD4 and CD8 T-lymphocytes from tumor immunized donors can transfer protective immunity across the blood-brain barrier into recipient rats which are tumor challenged intracerebrally.

Cytochrome p4501A1 gene (CYP1A1) and glutathione S-transferase mu gene (GSTM1) are involved in the metabolic activation or detoxification of environmental carcinogens including benzo[a]pyrene in tobacco smoke. Individuals with both Val/Val and C type of CYP1A1 (CYP1A1; Val/Val and CYP1A1; C) or homozygous null (-/-) genotype of GSTM1 gene (GSTM1; -/-) show increased susceptibility to lung cancer. The incidence of p53 gene mutations are related to the smoking index of the lung cancer patients. Therefore we determined genotypes of these enzymes and screened p53 gene mutations in 123 non-small cell lung cancer (NSCLC) patients. p53 gene mutations were found in 35% (43/123) of the patients. The incidence of p53 gene mutation CYP1A1; Val/Val (60.0%), CYP1A1; C (50.0%) tended to be higher than those of CYPIAI; Ile/Ile and Ile/Val (40.4%) or CYP1A1; A and B (40.5%). We conclude that the incidence of the p53 mutations does not seem to be significantly affected by only CYP1A1 or GSTM1 polymorphisms in lung cancer patients.

Using a multi-attribute approach, we and others have reported on the comprehensive health status of survivors of cancer in childhood, after completion of therapy. However, there is a dearth of information about health status and associated health-related quality of life (HRQL) in children and adults with cancer, during the treatment process; a deficit which this study was intended to redress. All children (n=18) receiving 'maintenance' chemotherapy for acute lymphoblastic leukemia (ALL) at a single institution were assessed prospectively through a single cycle of therapy. Patients, family members and health care professionals used global ratings, the health utilities index mark 2 (HUI2) and mark 3 (HUI3) classification systems, and detailed descriptions of temporary states of health, to provide independent assessments of health status at weekly intervals. Utility scores were derived for each comprehensive health state and for single attribute levels of the HUI2 system, and for the temporary health state descriptions. The classification of the subjects into the most appropriate temporary health states was challenging even for older children and some of the parents. The HUI instruments were used much more easily and produced highly comparable information. The most frequently affected attributes were pain, emotion and mobility/ambulation; in that order. The global (p=0.005) and specific morbidity burdens were, as predicted, greatest in the middle of the cycle of chemotherapy, reflecting the toxicity of steroid use. HUI2 global utility scores demonstrated moderate responsiveness with an intra-class correlation coefficient of 0.43. Cyclical administration of therapy, in particular corticosteroids, produces important changes in health status and HRQL in children with ALL. The HUI are valid and responsive systems for quantifying the burden of morbidity during the treatment of cancer in childhood. These are sensitive tools which can be used in a wide array of other circumstances to quantify changes in health status.