Functionally active toll-like receptor 3 on human primary and metastatic cancer cells.

Toll-like receptor 3 (TLR3) is a member of Toll-like receptors who recognize structurally conserved molecules derived from pathogens and trigger the immune response. To clarify if TLR3, is expressed in certain tumour cell lines and whether it is functional and what is the response of these cell lines to different concentrations of poly I:C treatment, we have screened SW480, SW620, FaDu and Detroit 562 cell lines using real-time PCR, flow cytometry and ELISA. We have shown that all these cell lines express TLR3 on mRNA and protein level but it is only functional in Detroit 562 cell line since only in these cells poly I:C treatment triggered the IL-6 secretion. In addition, poly I:C treatment inhibited cell growth and triggered up-regulation of IL-12p40, IL-8 and Il-1alpha in Detroit 562 cell line. By using annexin-V apoptosis detection kit, we have found that poly I:C triggers apoptosis in Detroit 562 cell line. We have found here that based on the results of TLR3 functionality there is a huge difference between FaDu and Detroit 562 cell lines which are of the same origin (pharynx) but FaDu is primary and Detroit 562 metastatic carcinoma. Our study also shows that Detroit 562 cell line could be a good model for cancer therapy research and development as it is responsive to TLR3 agonists which consequently drives it to apoptosis.

Gene expression profiling of Nm23-H2 overexpressing CAL 27 cells using DNA microarray.

Nm23-H1/NDPKA and Nm23-H2/NDPKB belong to a large family of NDP kinases, group of structurally and functionally closely related enzymes. The Nm23/NDPs are known to catalyse the transfer of terminal phosphates from ATP to other NTPs and dNTPs. Besides their role in the maintenance of the cells NTP pool the nm23 genes/proteins are known to have additional different biological functions, the most important being its metastasis suppressor activity. The complete picture of roles, actions and targets of nm23 genes/proteins is yet to be discovered. Our goal was to identify the downstream targets of Nm23-H2 by subjecting Nm23-H2 overexpressing CAL 27 cells (oral squamous cell carcinoma of the tongue) to microarray analysis. Using this powerful technology we identified genes, groups of genes and signalling pathways that could be clustered into several groups: apoptosis related genes, cell cycle and DNA damage, TGFbeta (transforming growth factor beta) signalling pathway and related molecules, WNT signalling pathway, differentiation and epithelial structural and related molecules, cell adhesion, metalloproteinases and their inhibitors, vesicular transport related molecules, proteasome associated, ubiquitin mediated proteolysis and several metabolic pathways. Based on these results we suggest that nm23-H2 might have an important role in oral squamous cell carcinoma which is to be confirmed by future studies.

Carcinomas of the cervix and corpus uteri in humans: stage-dependent blood levels of substance(s) immunologically cross-reactive with insulin.

Inasmuch as the elevated levels of substance(s) immunologically cross-reactive with insulin (SICRI) in a diabetic woman with carcinoma of the corpus uteri decreased following the surgical removal of the uterus and ovaries, 80 women with cervical carcinomas of various stages and 70 women with carcinomas of the corpus uteri of various stages were screened for the levels of SICRI and C-peptide. The levels of SICRI in the second, third, and fourth stages of the cancers were elevated (up to six times above the normal levels of immunoreactive insulin) and stage-dependent. The levels of C-peptide, which are related to the insulin-secreting activity of pancreatic beta-cells, were normal and independent of the stage of cancer.

Growth and treatment of Ehrlich tumor in mice with alloxan-induced diabetes.

Hypoglycemia and hypoinsulinemia accompanied i.p. or i.m. growth of the Ehrlich tumor in CBA/H and BALB/c mice. Simultaneously, insulin accumulated in the ascitic fluid of tumor-bearing mice. In hosts rendered diabetic by means of alloxan, the tumor decreased the blood glucose almost to the level seen in nondiabetic mice. Tumor growth was retarded in diabetic hosts, but cells from such tumors, transplanted into secondary diabetic recipients, grew faster than in their primary diabetic hosts, similarly to "nondiabetic" tumor cells growing in nondiabetic hosts. This phenomenon of "adaptation" of the tumor to the diabetic state was prevented if diabetic tumor-bearing mice were daily treated with insulin. The tumor did not grow in all diabetic recipients; the frequency of takes correlated with severity of the diabetes, i.e., with the dose of alloxan given to induce it. The greater the dose, the less mice accepted the tumor. Insulin injection into diabetic tumor-bearing mice promoted the tumor growth. Simultaneous treatment of diabetes and the tumor afforded the best antitumor effect.

Loss of heterozygosity and protein expression of APC gene in renal cell carcinomas.

This study evaluated the potential contribution of the APC gene to malignant transformation in patients with renal cell carcinoma. We tested 36 human renal cell carcinoma samples and 18 adjacent normal kidney tissues for the expression of APC protein, both wild and truncated types, by western blot using antibodies that recognize either the carboxy or the amino epitope of the APC protein. The same tumor samples together with autologous peripheral blood were also analyzed at the DNA level. Using specific oligonucleotide primers for exons 11 and 15, gene instability was followed by polymerase chain reaction/loss of heterozygosity (LOH) (on the basis of restriction fragment length polymorphism). Molecular data were also compared to pathohistological diagnosis, TNM stage, and patient's age using multivariate statistical methods. All normal renal tissues revealed expression of the wild-type APC protein. Neither wild nor mutant type proteins were found in 36% (13/36) of tumor samples; the rest of tumor tissues expressed the wild-type protein (312 kDa). Mutated APC protein, with a molecular weight of 117 kDa, was found in only one tumor sample. From 36 tumor samples 16 (44.4%) were informative for RsaI exon 11 polymorphic site, while only half of these (8/16) demonstrated LOH. From 13 tumor samples that had no detectable protein product by western blot analysis eight were homozygous for the exon 11 polymorphism and were tested for another polymorphic site, MspI/exon 15. The overall proportion of LOH cases for both polymorphisms tested was 52.9% (9/17). Pathohistological diagnosis and molecular data showed no correlation. However, multivariate analysis determined a stage strong positive correlation of age and TNM with the presence of LOH and the absence of the wild-type APC protein. Out results suggest that the APC tumor suppressor gene plays a role in renal carcinogenesis. Alterations in this gene are responsible for tumor evolution and progression, but cannot be considered as a first event in tumor initiation.

Squamous cell lung carcinomas: the role of nm23-H1 gene.

This analysis of 32 pairs of human squamous cell lung carcinomas and normal matched control DNA demonstrates that loss of heterozygosity (LOH) is infrequent at the nm23-H1 locus, affecting only 2 of the 18 informative cases. Both LOH cases were in the tumor stage IIIA. One tumor was of poor and the other of moderate histological grade. These and an additional 34 tumor samples were also analyzed immunohistochemically for the presence of nm23-H1 protein. Of the 66 cases tested for the presence of nm23-H1 protein 54 were negative. Eight samples exhibited up to 35% positive cells (with weak immunostaining intensity) and four between 35% and 70% (moderate immunostaining intensity); no sample showed more than 70% positive cells. Noncancerous lung parts contained no nm23-H1 protein. nm23-H1 expression was independent of TNM stage, grade, tumor size, and patient's survival. Two samples with LOH were negative for nm23-H1 protein. We therefore conclude that neither loss of heterozygosity of the nm23-H1 gene nor the intensity of specific protein expression are related to squamous cell lung carcinoma development and progression.

The expression and role of insulin-like growth factor II in malignant hemangiopericytomas.

Hemangiopericytoma is a rare soft tissue tumor originating from contractile pericapillary pericytes. To address the issue of molecular genetic events that participate in genesis and progression of hemangiopericytoma we analyzed insulin-like growth factor (IGF) II and IGF I receptor in 29 tumors collected from a human tumor bank network. Seven of these tumors were associated with severe hypoglycemia; six were retroperitoneal and one was located in the leg. Of 22 tumors tested 12 (54.5%) exhibited IGF II mRNA, while almost 90% (17 of 19) of hemangiopericytomas exhibited IGF I receptor mRNA. Sera from some patients whose tumors expressed IGF II mRNA contained elevated levels of IGF II. Removal of the tumor eliminated most of the IGF II immunoreactivity from the sera. The potential role of IGF II as a growth-promoting factor was examined on three malignant primary hemangiopericytoma cell cultures. Extracellular addition of IGF II significantly enhanced cell proliferation in a dose-dependent manner. Antisense oligodeoxynucleotides that specifically inhibit IGF II mRNA, at a concentration of 40 or 80 micrograms/ml, inhibited the growth of hemangiopericytoma cells significantly, by 40%. Simultaneous administration of antisense deoxyoligonucleotides to both IGF II and IGF I receptor inhibited tumor cell proliferation by even 80%. Our data suggest that tumor cells produce IGF II, and that this in turn stimulates their proliferation by autocrine mechanisms.

Natural zeolite clinoptilolite: new adjuvant in anticancer therapy.

Natural silicate materials, including zeolite clinoptilolite, have been shown to exhibit diverse biological activities and have been used successfully as a vaccine adjuvant and for the treatment of diarrhea. We report a novel use of finely ground clinoptilolite as a potential adjuvant in anticancer therapy. Clinoptilolite treatment of mice and dogs suffering from a variety of tumor types led to improvement in the overall health status, prolongation of life-span, and decrease in tumors size. Local application of clinoptilolite to skin cancers of dogs effectively reduced tumor formation and growth. In addition, toxicology studies on mice and rats demonstrated that the treatment does not have negative effects. In vitro tissue culture studies showed that finely ground clinoptilolite inhibits protein kinase B (c-Akt), induces expression of p21WAF1/CIP1 and p27KIP1 tumor suppressor proteins, and blocks cell growth in several cancer cell lines. These data indicate that clinoptilolite treatment might affect cancer growth by attenuating survival signals and inducing tumor suppressor genes in treated cells.

Nm23-h1 protein in oligodendrogliomas.

We examined nm23-H1 protein levels in human oligodendrogliomas by immunohistochemistry. This class of brain tumor does not form spontaneous metastases, but its progression from benign (oligodendroglioma) toward malignant phenotype (oligodendroglioma transitionale and glioblastoma oligodendrogliale) can be followed. Two types of tumors, ODG-II and ODG-T, were highly positive for nm23 protein. However, there was no clear correlation between the extent of protein expression and tumor aggressiveness. No nm23 protein was detected in nonproliferative normal brain tissues and was found in only a few ODG-I specimens. As cell proliferation becomes more pronounced (OGD-II, ODG-T), nm23 protein becomes detectable in almost all samples. However, of the glioblastoma oligodendrogliale samples examined, 76% were negative for nm23-H1 protein. suggesting a change in nm23-H1 gene expression with increasing neoplastic progression. Our findings are in contrast to a proposed role of nm23-H1 protein as a tumor metastasis suppressor and support that it cannot serve as a reliable prognostic tumor indicator in all cases. However, our findings may contribute to a better understanding of glial tumor development and improve the accuracy of tumor diagnosis.

Presence of oestrogen receptors on target cells and antiproliferative activity of estramustine phosphate: positive correlation for human tumours in vitro.

Incubation with estramustine phosphate for 24 h inhibited DNA, RNA and protein synthesis in primary cultures of human kidney, mammary, prostatic, cervical and endometrial carcinoma. Not only the presence, but also the concentration of oestrogen receptors correlated with estramustine phosphate effects on tumour cell proliferation.

Habituation of a mammary aplastic carcinoma on diabetic conditions.

Hypoglycemia and hypoinsulinemia accompanied the i.m. growth of mammary aplastic carcinoma in CBA mice. In hosts rendered diabetic by means of alloxan, the tumor decreased blood glucose levels to almost the level seen in non-diabetic mice. Tumors maintained in diabetic mice grew faster after each subsequent transplantation into diabetic mice, and we noted increased incorporation of 3H-thymidine into DNA of these tumor cells. The observed proliferation enhancement of mammary aplastic carcinoma maintained in diabetic mice is caused by de novo insulin and glucagon synthesis, apparently by the tumor cells themselves.

Collision tumour in the pelvic cavity: rectal leiomyosarcoma and prostate adenocarcinoma.

We report a rare and, to our knowledge, as yet undescribed type of collision tumour - rectal leiomyosarcoma and prostate adenocarcinoma. Our study also provides the first data on molecular alterations [polymerase chain reaction/loss of heterozygosity (LOH) analysis] of the APC, NF-1, DCC, p53, nm23-H1 and BRCA-1 genes in the two components of the collision tumour. None of the genes examined in this study expressed LOH in the prostate carcinoma component of the collision tumour. By contrast, in the leiomyosarcoma component, LOH was found at the DCC and p53 genes, proving that these two tumours did not arise from the same stem cell but represent two different neoplastic growths.

Loss of heterozygosity of the nm23-H1 gene in human renal cell carcinomas.

This study evaluates the potential contribution of the nm23-H1 gene to malignant transformation in patients with renal cell carcinoma. Using specific oligonucleotide primers for the nm23-H1 microsatellite repetitive sequence, gene instability was followed by polymerase chain reaction/loss of heterozygosity assay on 54 tumor specimens and the corresponding normal tissue samples. We also determined, immunohistochemically, the relative concentration and localization of the nm23-H1 protein product. From 77.7% informative cases, DNA from 6 tumors exhibited loss of heterozygosity, regardless of the tumor stage (TNM). Out of 39 samples analyzed, 30 were negative for Nm23-H1 protein, while the others were only slightly positive. No correlation with tumor stage was found. Normal renal tissue was also negative for this protein. Our results provide the evidence for loss of heterozygosity, followed by means of microsatellite tandem-repeat polymorphism, at the nm23-H1 locus in renal cell carcinoma. However, since no correlation was found between the tumor stage or metastatic potential on the one hand, and allelic loss and specific protein expression on the other, it seems that nm23-H1 does not play a key role in the invasiveness of this tumor type.

The role of insulin-related substance in Hodgkin's disease.

An insulin-related growth-promoting substance was detected in the serum of a patient with Hodgkin's disease who suffered from severe hypoglycaemia, as well as in the supernatant of homogenized spleen tissue of the same patient. Low concentrations of this substance enhanced DNA synthesis of short-term-cultured spleen tumour cells obtained from the same patient, while the addition of anti-insulin antiserum interfered with that effect. Moreover, the preincubation of this insulin-related substance with the anti-insulin antiserum abrogated its stimulatory effect on tumour cell proliferation. Both insulin and the insulin-related substance bound to patients splenocytes to a similar extent. The data suggest that the insulin-related substance, found in this particular case of Hodgkin's disease, plays a role in tumour progression by an autocrine mechanism.

Molecular pathology of hemangiopericytomas accompanied by severe hypoglycemia: oncogenes, tumor-suppressor genes and the insulin-like growth factor family.

Relatively little is known about molecular genetic events that participate in the genesis and progression of hemangiopericytoma. In this study, we describe two cases of hemangiopericytoma accompanied by severe hypoglycemia. Tumor cells from patient 1 exhibited insulin-growth factor I (IGF I) and insulin-like growth factor I receptor (IGF IR) mRNA transcripts. Tumor cells from patient 2 exhibited IGF II, IGF IR and IGF binding proteins 1-3 mRNA. Serum from patient 2 contained IGF II, mostly in a large molecular form ("big" IGF II); the IGF II level did not change after the tumor removal. The presence of IGF IR in tumor cells was confirmed by immunoprecipitation with antibodies that recognize human IGF IR subunit (visualized as a 460-kDa band). The hemangiopericytoma cells derived from patient 1 expressed 210000 IGF I receptors/cell. Specific binding of IGF I to the tumor cell membrane fraction was higher in tissue from patient 1, while the tissue of patient 2 showed relatively low IGF I binding. In contrast, IGF II binding was much higher in tissue from patient 2. Both tumor tissues showed positive immunostaining for c-Jun; one tumor showed strong immunostaining for c-Myc, H-Ras and p53, while the other exhibited strong reaction with H-Ras antibodies only. No loss of the heterozygosity at the genes APC, NFI and nm23-H1 loci in tumor tissue obtained from patient 1 was found. In effect, our results suggest multiple molecular genetic changes in hemangiopericytoma -- activation of some oncogenes and the IGF growth factor family. IGF ligands together with IGF IR could be responsible for hypoglycemia and perhaps the transformed phenotype.

PCR amplification of DNA from stained cytological smears.

DNA from archival Papanicolaou stained smears was successfully amplified using the polymerase chain reaction (PCR) to see if it could be used for retrospective genome studies such as detection of the presence of human papilloma virus (HPV) and changes in p53 gene expression. DNA was isolated and purified by treatment with proteinase K, phenol/chloroform, and isoamyl alcohol. Segments of the human beta actin and TGF beta 1 gene were amplified by PCR. Of all stains used in the preparation of Papanicolaou smears, only eosin was detectable as a greenish band in ethidium bromide treated DNA gels under ultraviolet illumination.

Evidence for a role of EGF receptor in the progression of human lung carcinoma.

Sixty-three primary lung carcinomas were examined immunohistochemically for the presence of epidermal growth factor receptors (EGF-R). Of these tumors 35 (55.5%) were positive for EGF-R. A positive correlation between overexpression of EGF-R, on the one hand, and high metastatic rate, poor tumor differentiation and high rate of tumor proliferation, on the other hand, was found. From one tumor with an extremely high amount of EGF-R a primary cell culture was established. The addition of anti-EGF-R serum to this culture decreased cell proliferation. Our results support the evidence for a positive relationship between overexpression of EGF-R and invasiveness, poor differentiation and high growth rate of tumor cells. The blockade of EGF-R with specific antibodies suppressed tumor cell proliferation, suggesting a role of EGF-R in tumor progression.

The p53 and nm23-H1 genes are not deleted in oral benign epithelial lesions.

In an effort to identify genetic changes that may be the early hallmarks of epithelial cell overproliferation, we searched for p53 and nm23-H1 allelic deletions in oral benign epithelial lesions. In the study group were 25 benign epithelial lesions (lichen planus--17; leukoplakia--8; recurrent aphthous ulcers--2; one specimen diagnosed as benign migratory glossitis). Among 21 samples analysed for exon 4 (p53 gene) LOH, only 6 were informative, with no loss of either allele. OF 23 samples tested for LOH at intron 6 of p53 gene, 8 were informative, again with no presence of LOH. For nm23-H1 gene, the analysis was performed on a total of 24 cases. Of them, 16 were informative, however, none exhibited LOH at this locus. In conclusion, whereas the presence of gross gene alterations (LOH) would have been definitive evidence for the involvement of p53 and/or nm23 in the hyperproliferation process, the absence of LOH does not exclude the presence of either smaller mutations, altered regulation of normal gene, or dysfunction at the level of wild type protein. Alternatively, p53 and nm23-H1 may have no relation to oral lesion formation, and cannot presently be considered as an early step in benign, tissue transformation.

Human lung cancers growing on extracellular matrix: expression of oncogenes and growth factors.

The goal of this study was to evaluate the extracellular matrix (ECM) as a model for growing human lung cancers and to study the feasibility of its application for cellular and molecular studies of tumor biology. Bovine corneal endothelial cell ECM coated dishes were evaluated as a growth substrate for tumor cultures. Growth success, morphology and oncoprotein/growth factor expression for 74 different lung cancers (adenocarcinoma, epidermoid carcinoma and small cell carcinoma) were compared after seeding fresh surgical explants onto bovine corneal endothelial cell ECM and plastic culture substrate. Nineteen out of 74 tumors (26%) plated on ECM demonstrated measurable growth. Growth on ECM was superior to growth on plastic for the lung tumors. All 19 tumor cultures showed malignant morphology and functions. They were examined under the light microscope, and in all cases pre- and post-cytology confirmed malignancy. Tumor cells seeded on ECM retained their malignant phenotype in comparison to tumors grown on plastic. Several oncoproteins (c-myc, c-Ha-ras, c-erbB-2) and growth factors/receptors (EGF, EGF-R, TGF alpha) were immunostained. These analyses were performed immediately after disaggregation of tumor cells obtained surgically and after seeding on ECM or plastic. Strong expression of oncoproteins/growth factors was detected in tumor cells immediately after surgery or when the cells were plated on ECM. On the other hand, moderate or no expression was observed in the same type of cells on plastic.

Effect of extracellular NADH on human tumor cell proliferation.

We investigated the antiproliferative effects of extracellular nicotinamide adenine dinucleotide against human malignant CaCo-2 (colon carcinoma), Hep-2 (laryngeal carcinoma), MCF-7 (breast carcinoma), CaSki (cervix carcinoma) cell lines, as well as against murine fibrosarcoma and normal human embryonal fibroblast (HEF). NADH was very potent in the growth inhibition of murine fibrosarcoma and human Hep-2 cells, regardless of the dose applied. During the observed period (4 or 5 days) only one dose of NADH was sufficient in reducing the growth rate for up to 92%. It had no effect on the growth of other cell lines tested. The identification of DNA-fragmentation and p53 and Ki-67 genes expression suggest that the mechanism of NADH action is different from disregulation of genes considered as check-points in cell cycle.