Siegina G. Evers

Mutational activation of the K-ras oncogene. A possible pathogenetic factor in adenocarcinoma of the lung.

To define the role of cellular oncogenes in human cancers, we studied the prevalence of mutational activation of ras oncogenes in untreated non-small-cell lung cancer. Genomic DNA was extracted from 39 tumor specimens obtained by thoracotomy and was examined for activating point mutations in codons 12, 13, and 61 of the H-ras, K-ras, and N-ras genes. A novel, highly sensitive assay based on oligonucleotide hybridization following an in vitro amplification step was employed. The K-ras gene was found to be activated by point mutations in codon 12 in 5 of 10 adenocarcinomas. Two of these tumors were less than 2 cm in size and had not metastasized. No ras gene mutations were observed in 15 squamous-cell carcinomas, 10 large-cell carcinomas, 1 carcinoid, 2 metastatic adenocarcinomas from primary tumors outside the lung, and 1 small-cell carcinoma. An approximately 20-fold amplification of the unmutated K-ras gene was observed in a tumor that proved to be a solitary lung metastasis of a rectal carcinoma. We conclude that mutational K-ras activation may be an important early event in the pathogenesis of adenocarcinoma of the lung but that amplification of ras genes or mutational activation of H-ras or N-ras does not play a major part in non-small-cell lung cancer.

A Rapid and Simple Procedure for the Routine Detection of ras Point Mutations in Formalin-Fixed, Paraffin-Embedded Tissues

The use of the polymerase chain reaction (PCR) to detect specific DNA sequences in small amounts of tissues or cells has become a widespread tool in the field of molecular biology. With the better understanding of the clinical significance of oncogene activations in human tumors, the application of PCR in a routine setting is rapidly gaining importance. We have developed a rapid and simple procedure for the detection of mutated ras oncogenes in routinely fixed, paraffin-embedded tissue samples. DNA is isolated from three 10 μm tissue sections by incubation with a nonionic detergent and proteinase K, and can be directly used for amplification by PCR. The amplified DNA fragments are then dot-blotted onto nylon membranes and are hybridized to radioactively labeled oligodeoxynucleotides, specific for each of the mutated ras sequences. After a selective washing procedure, only fully matched oligodeoxynucleotides remain bound to the membrane, thus revealing the nature of the sequences that were present in the starting material. With this method, the detection of point mutations in ras genes can be performed in a routine setting, and the results of the analyses can be available in as few as 3–4 days.

Combined endocrine therapy and chemotherapy of mouse mammary tumors.

Abstract Hormone-responsive mammary tumors of GR mice were treated with tamoxifen, cyclophosphamide, or with both drugs combined. Tamoxifen alone or cyclophosphamide alone caused inhibition of tumor growth, but more growth inhibition was obtained with the combined therapy.

Outgrowth of grafts containing different ratios of hormone-dependent and independent mouse mammary tumor cells

The growth properties of grafts composed of different mixtures of hormone-dependent and independent GR mouse mammary tumor cells were investigated. Grafts containing only hormone-dependent mammary tumor cells yielded tumors in 4--6 weeks in estrone plus progesterone treated castrated mice, but not in castrated mice that were not treated with hormones. Grafts containing only autonomous mammary tumor cells yielded tumors in about 2 weeks in both hormone-treated and untreated castrated mice. Grafts containing mixtures of hormone-dependent and autonomous cells yielded tumors in 2 weeks both in hormone-treated and untreated castrates, even when the grafts contained predominantly hormone-dependent cells (up to 90%). These results indicate that the growth behavior of heterogeneous mammary tumor grafts that contain more than 10% autonomous cells, is essentially determined by these cells. Growth of the autonomous cells is not markedly affected by the presence of hormone-dependent cells in the grafts.

Different int-1 region DNA rearrangements within different zones of a single mouse mammary tumor

Fragments were taken from separate parts of hormone-dependent (HD) primary GR mouse mammary tumors and serially transplanted in estrone plus progesterone treated or hormonally untreated castrated mice. The transplants were examined with respect to int-1 DNA rearrangement, proviral integrations of the murine mammary tumor virus (MMTV), and estrogen and progesterone receptor content. One of the fragments (b) taken from the primary tumor of line TSI 96 produced transplants that showed int-1 rearrangement in one allele and also MMTV proviral integrations not at the int-1 gene, whereas transplants from another fragment (a) only had the normal germ-line int-1 arrangement and no extra MMTV provirus. These respective genotypes were retained when the tumors became hormonally independent during further transplantations. The results indicate that int-1 rearrangement was not present in the originally transformed cell but occurred in a HD cell during growth of the tumor. Furthermore they indicate that loss of hormonal dependence in GR mammary tumors is due to a mutational event, unrelated to int-1 rearrangement.

Hormonal regulation of mouse mammary tumor growth

In view of reports that human breast cancer cells secrete growth factors that can replace estradiol in sustaining tumor growth [1], we have investigated whether hormone independent (HI) GR mouse mammary tumors can sustain growth of estrogen-depleted hormone dependent (HD) tumors. HD GR mammary tumor TSl 106 was grafted subcutaneously in the right flank of estrone plus progesterone treated castrated (020 X GR)F1 mice. After 2 weeks the estrone treatment was stopped and the mice received 50, 100 or 150 mg HI GR mammary tumor TSl 104 in the left flank. However, the regression of the HD tumor due to estrone depletion was not prevented or retarded by the HI grafts. In other experiments we investigated integrations of mouse mammary tumor virus (MMTV) proviral DNA in the DNA of GR mammary tumors. We could demonstrate the presence of two cell populations in tumor TSl 96, both HD but differing in MMTV DNA integration events. Our data indicate that exogenous integrations of MMTV proviruses can take place in mouse mammary tumor DNA without loss of hormone dependency of the tumors. Like in GR/Mtv-2+ mice, mammary tumor transplants differing in MMTV proviral integrations are also observed in 020/Mtv-2+ mice.

Chemotherapy and hormonal therapy of mouse mammary tumors.

Single Cisplatin (Platinol) injections i.p. at 10 mg/kg dose level (LD10) caused a marked growth inhibition of both hormone dependent and independent GR mouse mammary tumors (reduction in tumor volumes of 86 and 85%, respectively). However this treatment caused severe toxic side effects including loss of mouse body weight and deaths of some of the animals. When Cisplatin (pure) was administered s.c. in a single cholesterol pellet, no such toxic effects were observed even when Cisplatin levels up to 30 mg/kg were applied. The latter dose caused 29% reduction in tumor volume of hormone independent tumors. However this inhibitory effect was temporary, since regrowth of the hormone independent tumors started again 8 days after application of the single Cisplatin pellet. These results show that continuous Cisplatin administration in pellets s.c. is less toxic than injection i.p., but that the inhibition of mammary tumor growth is also less marked.