Germline MSH2 and MLH1 mutational spectrum including large rearrangements in HNPCC families from Poland (update study).

Germline mutations in the DNA mismatch repair genes MSH2 and MLH1 account for a significant proportion of hereditary non-polyposis colorectal cancer (HNPCC) families. One approach by which development of an efficient DNA-testing procedure can be implemented is to describe the nature and frequency of common mutations in particular ethnic groups. Two hundred and twenty-six patients from families matching the Amsterdam II diagnostic criteria or suspected HNPCC criteria were screened for MSH2 and MLH1 germline mutations. Fifty different pathogenic mutations were found, 25 in MSH2 and 25 in MLH1. Twenty-four of these had not previously been described in other populations. Among our 78 families with MSH2 or MLH1 mutations, 54 (69.2%) were affected by recurrent mutations including 38 found at least twice in our own series. Two of the most frequent alterations were a substitution of A to T at the splice donor site of intron 5 of MSH2 and a missense change (A681T) of MLH1 found in 10 and eight families, respectively. Among large deletions detected by the multiplex ligation-dependent probe amplification assay, exon 9 deletions in the MSH2 gene were found in two families. Our results indicate that a screening protocol specific for the Polish population that is limited to the detection of all reported mutations will result in the identification of the majority of changes present in MLH1 and MSH2 genes in Polish HNPCC kindreds.

Family with Li-Fraumeni syndrome and no evidence of a germline mutation of the p53 gene or chromosomal aberrations.

Li-Fraumeni syndrome is a rare autosomal, dominant trait of diverse types of cancers in children and young adults, with a predominance of soft tissue sarcomas, osteosarcomas, brain tumours, adrenocortical and breast carcinomas, as well as leukaemias. We present a family with an unusual cancer history fulfilling the criteria of Li-Fraumeni syndrome. Mutational analysis of the p53 gene in constitutional DNA of several affected members of the family did not show any germline p53 defect. Cytogenetic studies did not reveal any structural aberrations.

Screening for germline p53 mutations in pediatric and adult patients of high-risk groups in Poland.

Germline mutations of the p53 gene lead to cell transformation in various tissues. Such a complex cancer phenotype makes it difficult to recognize the carriers of the defective allele. Several studies undertaken to identify high-risk groups found germline p53 mutations in familial cancer aggregations and in patients with multiple tumors. We screened 189 pediatric and 48 adult patients. The high-risk groups comprised 41 patients with a family history of cancer and 35 with multiple neoplasms. Furthermore, 124 tumors were screened for somatic mutations. p53 exons 2 to 11 were analyzed by polymerase chain reaction and single strand conformation polymorphism (PCR-SSCP) followed by direct sequencing of abnormal DNA fragments. No germline p53 mutations were found and somatic mutations were detected in 5 of 59 sarcomas, globally, in 8 of 124 tumors. In conclusion, in Poland, p53 alterations do not seem very important for the predisposition to malignancy and development of sarcomas.

Local low-dose IL-2 therapy.

Interleukin-2 (IL-2) is a powerful drug for treating cancer. However, it is only powerful if it is properly applied. That is, IL-2 should be applied at the tumor site, because at the transition of normal and malignant tissue are the tumor infiltrating cells. These should be activated by IL-2. Local application implies that IL-2 can be used in relatively low doses. It is becoming clear that even a single injection of IL-2 can cure cancer. IL-2 can also enhance the therapeutic effects of irradiation and Cisplatin. Locally applied IL-2 therapy is virtually non-toxic.

Effect of local interleukin-2 treatment on spontaneous tumours of different immunogenic strength.

Transplantable tumour lines established from spontaneous tumours of BALB/c, CBA, and DBA/2 mice displayed different immunogenic strength. This report describes tumour susceptibility to interleukin-2 (IL-2) therapy in relation to tumour immunogenicity. The following tumour lines were used: X5, X6, and X9 mammary tumours of DBA/2, BALB/c, and CBA origin respectively, X7 carcinoma of BALB/c and X18 papilloma of CBA mice. Two spontaneous tumours of long transplantation history, SL2 lymphoma (SL2) of DBA/2 and Madison lung carcinoma M109 (M109) of BALB/c origin, were used as control systems. Experimental mice were transplanted with different inocula of tumour cells at day 0; treatment with IL-2 was initiated on days 1-3 or delayed until day 10 and consisted of daily injections of low doses of 5000 or 20,000 U/mouse given five times a week for a period of 3 weeks. Treatment of SL2 (2 x 10(4) cells injected i.p.) consisted of i.p. injections of 5000 or 20,000 U IL-2/mouse given on days 10-14 after tumour transplantation. IL-2 therapy of SL2-bearing DBA/2JIco mice resulted in a significant proportion of cures; however, no response to IL-2 treatment was achieved in SL2-bearing DBA/2CrIiw mice. BALB/c mice with the i.p. transplant of M109 responded to IL-2 treatment with 40% increase in lifespan. The low-dose IL-2 therapy of the five spontaneous tumours resulted, in general, in transient growth inhibition of the i.m. transplants of lines X5, X6, and X7 provided that IL-2 was administered locally. The therapeutic effect depended on the number of transplanted tumour cells, the best results being achieved at cell numbers close to the dose-inducing tumour growth in 50% of animals. We found that the spontaneously arising tumours responding to IL-2 treatment were all slowly growing and immunogenic (X6 and X7) or might have viral association (X5) and, as such, might express foreign antigens. The data suggest a correlation between tumour immunogenicity and the therapeutic effect. However, IL-2 can still exert some effect against tumours with negligible immunogenicity.

Therapeutic potential of biological response modifiers against transplantable mouse tumors of spontaneous origin. II. Local interleukin 2 treatment of tumors of different immunogenic strength.

This report describes tumor susceptibility to interleukin 2 (IL-2) therapy in relation to tumor immunogenicity. The following lines recently established from spontaneous tumors were used: X5, X6, and X9 mammary tumors of DBA/2, BALB/c and CBA origin, respectively, X7 carcinoma of BALB/c and X18 papilloma of CBA mice. Two spontaneous tumors of long transplantation history, SL2 lymphoma (SL2) of DBA/2 and M109 Madison lung carcinoma (M109) of BALB/c origin, were used as control systems. Mice were transplanted with different inocula of tumor cells at day 0; IL-2 treatment was initiated on day 1-3 or 10 and consisted of daily injections of 5000 or 20,000 IU/mouse given 5 times a week for 3 weeks. SL2 (i.p. - 2 x 10(4) cells) treatment consisted of i.p. injections of 5000 or 20,000 IU IL-2 given on days 10-14. IL-2 therapy of SL2 bearing DBA/2JIco mice resulted in the significant proportion of cures; however, no response to IL-2 treatment was achieved in SL2 bearing DBA/2Crliw mice. BALB/c mice with the i.p. transplant of M109 responded to IL-2 treatment with 40% increase in lifespan. The low-dose IL-2 therapy of the 5 recently established tumors resulted, in general, in transient growth inhibition of the i.m. transplants of line X5, X6, and X7 provided IL-2 was administered locally. The therapeutic effect depended on the number of transplanted tumor cells, and the best results being achieved at cell numbers close to the dose inducing tumor growth in 50% of animals (TD50). We found that the tumors responding to IL-2 treatment were all slowly growing and immunogenic (X6 and X7) or might have viral association (X5) and as such might express foreign antigens.

Therapeutic potential of biological response modifiers against transplantable mouse tumors of spontaneous origin. I. Characterization of biological properties of tumor lines and preliminary data on response to cytostatic drugs and biomodulators.

Several transplantable lines were derived from spontaneous tumors of mouse strains of low and high cancer incidence (CBA, BALB/c and DBA/2), and eight of them were used for an evaluation of the therapeutic potential of BRM. The lowest number of cells inducing tumors was in most cases around 10(4) cells. In transplantation tests, tumors of CBA and DBA/2 mice were non-immunogenic, while tumors of BALB/c mice showed different level of immunogenicity. Transplantation of a small number of tumor cells in admixture with BCG resulted in no tumor growth of the immunogenic line X6 and no effect on take rate of non-immunogenic line X1. Sensitivity of studied tumor lines to BCNU, CY and ADR was moderate. Therapy of the tumors with postulan and CMA, two biomodulators already shown to be active against various experimental tumors, failed. The significant therapeutic response was observed in DBA/2 mice bearing i.p. transplant of lymphosarcoma X19 and treated with i.p. injections of CMA.

Introduction of the activated N-ras oncogene into human fibroblasts by retroviral vector induces morphological transformation and tumorigenicity.

The introduction of activated N-ras cDNA into normal diploid human skin fibroblast cell cultures using the retroviral vector pZIPneo results in a spectrum of morphologies ranging from near normal to, in rare instances, dense piled-up colonies of morphologically transformed cells. However, none of the clones isolated were transformed as assessed by growth on agar or tumorigenicity in nude mice. Introduction of both c-myc and N-ras oncogene cDNAs into normal skin fibroblasts failed to produce transformation as assessed by growth on agar and tumorigenicity in nude mice, although c-myc infection alone conferred immortality and the resultant doubly infected cell line was immortal. Using the same construct, activated N-ras cDNA was shown to transform immortalized human fibroblasts to tumorigenicity. However, immortalization per se was shown not to guarantee 'co-operation' with an activated N-ras gene to give malignant transformation. Although numerical and structural chromosome aberrations (clonal and non-clonal) were observed in some of the cell strains isolated after retroviral infection, these were not directly associated with viral infection, the presence of the oncogenes or with the morphologically transformed phenotype.

Antineoplastic activity of azacarbazoles. II. Effect of alpha-carboline and its derivatives on transplantable animal neoplasms.

The antineoplastic properties of alpha-carboline, alpha-carboline hydrochloride, alpha-carboline N-1 methyl iodide and c-6 substituted fluoro-, chloro, nitro- and phenyl-alpha-carboline were studied. None of the compounds proved to be active when tested against i.p. transplanted B16 melanoma or i.m. implanted Lewis lung carcinoma. In addition, alpha-carboline was assessed against i.p. inoculated plasmacytoma MP26 and colon carcinoma 26, and solid tumors of mammary carcinoma 16/C and Walker carcinosarcoma 256. Under conditions tested these neoplasms did not respond to alpha-carboline.

Antineoplastic activity of new linear hydrazine derivatives.

A series of hydrazine derivatives was tested for antineoplastic activity. Cyanoacetic acid hydrazide (I), cyanoacetic acid methylhydrazide (II) and N-thioamido-N'-cyano-acethylhydrazine (VI) appeared to be the most active agents against sarcoma 180, Ehrlich carcinoma and Nemeth Kellner lymphoma. The maximum tumor weight inhibition ranged from 70 to 90%. N, N'-bis-cyano-acethylhydrazine (IV) and N-isonicotinoyl-hydrazine (VIII) showed significant antitumor activity in Ehrlich carcinoma and Nemeth Kellner lymphoma systems. None of hydrazine derivatives were active against L1210 leukemia. The most active agents, I, II and VI were further evaluated in leukemia P388, melanoma B16, Lewis lung carcinoma and mammary carcinoma 16/C, and the agent VI was additionally tested in plasmacytoma MP26, colon carcinoma 26 abd Walker carcinosarcoma 256 systems. However, there was no effect with any agent or dose tested against any neoplasm.

Enzyme activity in mice with transplantable leukemia.

Activity of cobalt activated acylase, gamma-glutamyltransferase, leucylaminopeptidase and alanylaminopeptidase in serum and liver of mice with transplantable leukemias (L1210, L1210/ara-C, L1210/CH3-G, AKSL-4, plasmacytoma ADJ-PC-5) were determined. Adenosinotriphosphatase, 5'-nucleotidase and alkaline phosphatase were histochemically localized in lymphatic nodes and spleen. Among the investigated enzymes the rise in serum activity of cobalt activated acylase and gamma-glutamyltransferase was demonstrated. A substantial increase of leucylaminopeptidase and alanylaminopeptidase was shown in the liver. A decrease in the histochemical reactions of all the studied enzymes was observed.

Biological activity of 1,4-dihydropyridine derivatives.

Six new 1,4-dihydropyridine derivatives were evaluated in vitro for antimicrobial and cytotoxic effects and in vivo for antineoplastic activity. These compounds inhibited the growth of most of Gram-positive and Gram-negative bacteria at concentrations of 50 and 100 micrograms/ml. Concentrations effective against fungi were somewhat lower (25-50 micrograms/ml). The growth of mycobacteria was inhibited at concentrations of 3.1-25 micrograms/ml. Compound IV inhibited the growth of pathogenic mycobacteria including M. tuberculosis resistant to SM and INH at 3.1 or 6.2 micrograms/ml. In cytotoxicity assays, compound II, IV and V appeared the most active. However, none of the 1,4-dihydropyridine derivatives affected the survival time of mice with P388 and L1210 leukemias or melanoma B16. The growth of subcutaneous tumors of sarcoma 180 was inhibited by compounds I, III, IV and V. The effect was dose related.

Morphological changes in organs of mice induced by the combination of adriamycin and pustulan.

In CDF1 mice treated with the combination of pustulan (50 mg/kg) and a low dose of adriamycin (10 mg/kg), the profound changes were noted in the liver. They included both destructive lesions in hepatocytes and the proliferation of the RES. Similar morphological changes, though generally less pronounced, were observed in mice treated with a high dose of adriamycin (20 mg/kg). The myocardial lesions were found in mice treated with the high dose of adriamycin or the combination of pustulan with the low dose of adriamycin. Morphological changes in the myocardium, as opposed to those in the liver were not reversible. No considerable changes were seen in organs of mice treated with pustulan at a dose of 50 mg/kg.

Chemical, pharmacological and oncostatic properties of 5-(4'-hydroxybenzylidenoimino)-4, 6-diketo-4, 5, 6, 7-tetrahydropyrimidine-[4, 5-d]-3-methyl-isothiazole (compound IP-10).

Advanced preclinical studies on IP-10 preparation (4,6-diketo-4, 5, 6, 7-tetrahydropyrimidine-[4, 5-d]-3-methyl-isothiazole) were carried out. The drug was shown to be devoid of the irritating local effect, mildly toxic and hardly absorbing when administered per os. The toxic effect showed tendency toward cumulation. In the long-term exposure it did not affect either the elements of peripheral blood or parenchymatous organs. It exerted slight hypotensive effect on the circulatory system but only after intravenous administration. In relation to the smooth muscle organs and central nervous system, IP-10 was only slightly active. Weak effect of the compound was observed with bacteria and fungi. In the case of transplantable tumors, its activity was differentiated. It exerted a significant effect in relation to leukemia, melanoma B-16, Ehrlich carcinoma and Nemeth-Kellner lymphoma. As other isothiasole derivatives, IP-10 exhibits an interesting pharmacological, easy to render activity; particular attention should be paid to its oncostatis activity.

Effects of combined treatment of L1210 leukemia with adriamycin and pustulan.

The development of L1210 leukemia transplanted i.v. into CDF1 or BDF1 mice was not affected by pustulan. However, pustulan combined with adriamycin improved the final antitumor effect. The potentiation of the antitumor action was observed when the pustulan administration preceded by 15-8 days the adriamycin injection. The results indicated that the antitumor activity of the pustulan and adriamycin combination was proportional in a range of adriamycin doses (5-10 mg/kg) to the activity of adriamycin by itself. The enhanced tumor-inhibitory effect was accompanied by severe toxicity. Hence, the combination of pustulan with high doses of adriamycin i.e., 20 mg/kg, did not give further prolongation of the survival time of mice over that produced by adriamycin alone. The same relationships were observed when low (10(2) cells and high (10(6) cells) inocula of L1210 cells were grafted into both CDF1 and BDF1 mice.

Chemical and biological properties of 2-pyridyl-benzyl-carbinol.

Three isomers of pyridylbenzylcarbinol were synthetized and their chemical properties and biological activity were studied. Clear dependence between chemical structure and antitumor activity was observed. As expected most active appeared 2-pyridylbenzylcarbinol (alpha-carbinol). It showed significant antitumor activity against subcutaneous tumors of Ehrlich carcinoma. Nemeth-Kellner lymphoma and sarcoma 180. On a chronic treatment schedule tumor inhibition from 60 to 80% was induced. No activity was seen in L1210 and P388 models. In B16 melanoma the significant inhibition of tumor growth was obtained at 200 mg/kg/injection. Tumor inhibitory effect was also observed in Yoshida sarcoma. Depending on a dose and schedule the increase in lifespan from 45 to 147% was achieved. LD50 and MTD were determined and were 750 +/- 87.65 mg/kg and 557.8 mg/kg, respectively. Only slight suppressive effect of alpha-carbinol on immunological system was showed. It concerned mainly immunological reactions with cellular system involved, in which distinct suppressive effects were noted. Specific and nonspecific humoral reactivity was little or not at all affected by alpha-carbinol.