Genetic ablation: targeted expression of a toxin gene causes microphthalmia in transgenic mice.

Lineage-specific regulatory elements can be used to direct expression of a variety of genes to specific tissues in transgenic mice. If the hybrid constructs contain a gene encoding a cytotoxic gene product, then genetic ablation of a specific cell lineage can be achieved. We have generated six transgenic mice by introducing into fertilized eggs the mouse gamma 2-crystallin promoter fused to the coding region of the diphtheria toxin A-chain gene. Three of these mice and all the transgenic offspring analyzed were microphthalmic. The lenses of these mice displayed considerable heterogeneity: some were almost normal morphologically but reduced in size, whereas others were grossly aberrant and deficient in nuclear fiber cells. These studies indicate that programmed ablation of specific cell types can be stably transmitted through the germ line.

Cloning, sequence determination, and expression in transfected cells of the coding sequence for the tox 176 attenuated diphtheria toxin A chain.

DNA including the coding sequence for the A chain of the mutant diphtheria toxin tox 176 was cloned. The cloned mature A-chain coding sequence showed a G-to-A transition at nucleotide 383 as the only difference from the wild-type sequence. This resulted in replacement of the glycine at position 128 by aspartic acid in the predicted amino acid sequence. A eucaryotic cell expression plasmid, pTH1-176, was constructed in which the tox 176 A-chain coding sequence was attached to a truncated metallothionein promoter. The toxicity of this construct, compared with that of the corresponding wild-type diphtheria toxin A-chain plasmid, pTH1, was assessed after transfection into the human 293 cell line by an indirect transient expression assay (I. H. Maxwell, F. Maxwell, and L. M. Glode, Cancer Res. 46:4660-4664, 1986). For the same effect, 15- to 30-fold more pTH1-176 than pTH1 was required, a result consistent with previous in vitro estimates of the diminished activity of the tox 176 A chain. Controlled expression of the cloned tox 176 A-chain coding sequence may provide a means of eliminating specific cell populations in an organism, for which purpose the wild-type diphtheria toxin A chain might prove too toxic.

Macrophage colony-stimulating factor complementary DNA: a candidate for gene therapy in metastatic melanoma.

BACKGROUND: At present, there is no highly effective treatment for metastatic melanoma. Innovative approaches aimed at inducing a more effective immune response against tumors have shown promising results in animal models. One approach involves the genetic modification of tumor cells so that they produce cytokines that stimulate an immune response. PURPOSE: The aim of this study was to determine the effectiveness of cytokine gene therapy for metastatic melanoma in a murine melanoma model. METHODS: B16F10 murine melanoma cells, which readily metastasize to the lungs, were transduced with a retroviral vector containing genes encoding neomycin resistance and human macrophage colony-stimulating factor (M-CSF). The presence of M-CSF messenger RNA in transduced cells was examined by coupled reverse transcription and polymerase chain reaction. Concentrations of soluble M-CSF in cell culture supernatants were determined by enzyme-linked immunosorbent assays (ELISAs). A clonal cell line, designated N+/CSF+, that expressed and secreted M-CSF was identified. Another clonal cell line, designated N+/CSF-, did not secrete M-CSF at levels detectable by ELISA. B16F10, N+/CSF-, and N+/CSF+ cells, individually or in combination, were injected intravenously or subcutaneously into C57BL/6 mice; we then evaluated the tumorigenicity and metastatic behavior of the cells, as well as the immune responses and survival of the mice. The immune responses assayed were the cytotoxic T lymphocyte (CTL) and peritoneal exudate cell (PEC) tumoricidal activities. RESULTS: Injection of B16F10 cells into the tail vein of C57BL/6 mice led to the establishment of lung metastases by week 2 and death by week 8. Injection of the N+/CSF+ or N+/CSF- cells led to the establishment of lung metastases that were detected at 2 and 3 weeks, respectively; however, these metastatic lesions were eliminated, and the animals had survival rates similar to those of the noninjected control mice. Injection of mice with a mixture of B16F10 and N+/CSF- cells resulted in the development of metastatic disease and 0% survival at 8 weeks, whereas mice that had been given an injection of a mixture of B16F10 and N+/CSF+ cells had an 80% survival rate at 8 weeks and survived at least two times longer (P = .007). The CTL and PEC tumoricidal activities in animals given an injection of N+/CSF+ cells suggested that monocytes and lymphocytes were responsible for the observed antitumor response. CONCLUSION: These findings suggest that the expression of M-CSF by genetically modified melanoma cells caused an effective antitumor immune response in host C57BL/6 mice and, thus, prolonged survival over that observed in the control mice.

Reduced gamma-cystathionase protein content in human malignant leukemia cell lines as measured by immunoassay with monoclonal antibody.

A murine hybrid cell line has been produced which secretes immunoglobulin G2b with specificity for human gamma-cystathionase (EC 4.2.1.15). The antibody has been iodinated and used in combination with quantitative immunoelectrophoresis in an assay which is capable of detecting as little as 1.5 ng enzyme protein. Human lymphoblastic leukemia cell lines CEM and Laz-221 contain undetectable enzyme protein, corresponding to their behavior as cysteine auxotrophs. In contrast, nonmalignant lymphoblastoid lines contain easily detectable enzyme protein which correlates with their behavior as cysteine prototrophs. Other malignant leukocyte cell lines contained detectable but variable amounts of enzyme protein, suggesting that the enzyme may be a useful marker of cellular differentiation.

Expression of the diphtheria toxin A-chain coding sequence under the control of promoters and enhancers from immunoglobulin genes as a means of directing toxicity to B-lymphoid cells.

Previous results have shown that cells can be killed by the expression of an introduced gene encoding diphtheria toxin A-fragment (DT-A) and that killing can be targeted using tissue-specific transcriptional regulatory elements. Here, we describe expression plasmids containing the DT-A gene linked with promoters and enhancers from immunoglobulin heavy chain or kappa-light chain genes. When these plasmids were transfected into cultured cells, DT-A was expressed in B-lymphoid cells but not detectably in HeLa cells or fibroblasts. A high specificity for B-cells was confirmed by assaying for luciferase reporter gene expression from a plasmid containing an analogous combination of immunoglobulin heavy chain regulatory elements. A plasmid containing an immunoglobulin-kappa promoter and enhancer was substantially less active in expressing DT-A in a pre-B-cell line than in B-lymphoma cells, suggesting the possibility of targeting DT-A expression to mature, malignant B-cells while sparing normal B-cell progenitors. By means of viral delivery vehicles, the constructs described might be applied in gene therapy for B-cell leukemias or lymphomas.

Regulated expression of a diphtheria toxin A-chain gene transfected into human cells: possible strategy for inducing cancer cell suicide.

As an alternative to directing plant or bacterial toxins to surface receptors, we are investigating the possibility of killing tumor cells by the expression of an exogenously introduced toxin gene (i.e., cell suicide). Tissue-specific gene regulatory elements might thus be exploited to achieve selective killing. To assess the feasibility of such an approach, we have transfected human cells (HeLa, B-lymphoblastoid, and 293 cells) with plasmids containing the diphtheria toxin A-chain (DT-A) coding sequence. The presence of the DT-A sequence lowered the level of transient expression of chloramphenicol acetyltransferase from a cotransfected plasmid, pSV2cat. This expression level in B-cells was further diminished by the inclusion of an immunoglobulin enhancer in the DT-A plasmid. In cotransfection experiments with a DT-A plasmid lacking an enhancer, chloramphenicol acetyltransferase expression was much more strongly inhibited in 293 cells (which express adenovirus E1A and E1B products) than in the other cell types; furthermore, the presence of the DT-A sequence eliminated recovery of G418-resistant 293 cell transformants after transfection with a plasmid containing the neo selectable marker. These results suggest that cell-specific regulatory mechanisms can be exploited to achieve selective cell killing by expression of an introduced toxin gene.

A Phase 1 study of high doses of aminopterin with leucovorin rescue in patients with advanced metastatic tumors.

We have conducted a Phase 1 study of aminopterin (AMT) with leucovorin (LV) in 17 patients. AMT was administered by bolus injection every 7 to 14 days in dosages from 25 to 425 mg/sq m. LV rescue was instituted at 24 hr and continued for 48 to 72 hr. At dosages above 50 mg/sq m, we observed nephrotoxicity defined as greater than or equal to a 25% increase in serum creatinine 24 hr after AMT administration, but its incidence was not strictly dose related. Urinary alkalinization and volume expansion appeared to reduce the incidence of nephrotoxicity. Nephrotoxic drug courses were associated with 24-hr plasma AMT levels [3.6 +/- 2.0 (S.D.) X 10(-6) M] which were significantly higher than nonnephrotoxic courses (1.6 +/- 1.0 x 10(-6) M) (p less than 0.05). In nonnephrotoxic courses, serum elimination pharmacokinetics appeared to be biphasic with a t1/2 alpha of 1.08 +/- 0.01 hr and t1/2 beta of 12.31 +/- 0.06 hr. Systemic toxicity (myelosuppression and mucositis) could be prevented in patients with impaired AMT clearance by the administration of LV at an increased dose rate. In several courses, systemic toxicity occurred in spite of apparently normal plasma clearance, suggesting that 24-hr plasma levels may not accurately reflect intracellular drug effects. Cytokinetic studies on bone marrow aspirates allowed determination of the rescue effect of LV and may prove useful in predicting marrow protection.

Autologous bone marrow transplantation in the therapy of small cell carcinoma of the lung.

Ten patients with small cell carcinoma of the lung were entered into a chemotherapeutic treatment program consisting of cyclophosphamide, vincristine, Adriamycin, and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea. Two courses of combination chemotherapy were administered to each patient followed by a third course with the same doses of drugs used on Course 2 but with autologous bone marrow transplantation given 24 to 48 hr after drug infusion. No differences could be detected between Courses 2 and 3 in terms of the magnitude, timing, or degree of myelosuppression. Serial bone marrow biopsies documented a progressive decline in granulocyte-macrophage colony-forming units in culture per mg bone marrow medullary core from 138 +/- 179 (S.D.) prior to chemotherapy to 7 +/- 11 after the marrow transplant recovery (p = 0.05). These data suggest that autologous bone marrow transplantation does not reduce the myelosuppression seen following the drugs used in this study at the dosages used. Autologous bone marrow transplantation may be useful only in the setting of marrow lethal therapy. Its usefulness in shortening recovery time from nonlethal therapy appears questionable.

Challenges and opportunities of the Internet for medical oncology.

PURPOSE: The internet, and in particular the world wide web (www), has a rapidly increasing potential to provide information for oncologists and their patients about cancer biology and treatment. A brief overview of this environment is given along with examples of how easily the information is accessed as a means of introducing the web page of the American Society of Clinical Oncology (ASCO), ASCO OnLine. METHODS: Oncology information sources on the www were accessed from the author's home using a 14.4 kbs modem, Netscape browser (Netscape communications Corp, Mountain View, CA), and the locations recorded for tabulation and discussion. RESULTS: Overwhelming amounts of oncology-related information are now available via the Internet. CONCLUSION: Oncology as a subspecialty is ideally suited to apply the newest information technology to traditional needs in areas of education, research, and patient care. Oncologists will increasingly act as information guides rather than information resources for patients and their families with cancer.

Targeted ablation of pituitary gonadotropes in transgenic mice.

LH, FSH, and TSH are heterodimeric glycoprotein hormones composed of a common alpha-subunit and unique beta-subunits. The alpha-subunit is produced in two distinct specialized cell types of the pituitary gland: gonadotropes, which synthesize LH and FSH, and thyrotropes, which synthesize TSH. We have demonstrated that 313 base pairs of the bovine-alpha subunit promoter direct expression of diphtheria toxin A chain specifically to the gonadotropes in transgenic mice. Animals carrying this transgene generally exhibit reproductive failure and lack of gonadal differentiation, consistent with gonadotrope ablation. Lack of gonadotrope activity was verified by RIA and immunohistochemical staining for LH. The phenotype of these transgenic mice is nearly identical to mice homozygous for the spontaneous mutation, hpg, which is due to a deletion in the gene encoding GnRH. Thyrotrope function was judged normal based on overall growth of the animals, appearance of their thyroids, T4 levels measured by RIA, and immunohistochemical staining for TSH. The ablation of gonadotropes but not thyrotropes suggests that separate cis-acting elements are necessary for expression of the alpha-subunit gene in these two cell types. Pituitary content of ACTH and GH was apparently normal, while PRL synthesis and storage were reduced. Thus, in a pituitary almost completely devoid of gonadotropes, most other pituitary functions were normal. This suggests that most pituitary cells are able to differentiate independently of terminal gonadotrope differentiation and can function in the absence of paracrine signaling provided by gonadotropes.

Dose limiting extramedullary toxicity of high dose chemotherapy.

Autologous marrow support may allow higher doses of myelosuppressive chemotherapy administration with the hope of improving tumor responses. The approach will be limited by the extramedullary toxicities of the drugs employed. This paper reviews some of these toxicities for selected drugs, and gives examples of techniques which could be used to reduce their incidence.

Activation of a diphtheria toxin A gene by expression of human immunodeficiency virus-1 Tat and Rev proteins in transfected cells.

Expression of a gene encoding the diphtheria toxin A (DT-A) fragment, controlled by tissue specific regulatory elements, has previously been used to kill selected cell populations. Here, we have examined the feasibility of controlling DT-A expression using regulatory systems from the human immunodeficiency virus (HIV-1) genome. Plasmids were constructed which express either DT-A or, as a model system, the luciferase (luc) reporter gene, under control of HIV-1 long terminal repeat (LTR) sequences (-167 to +80). While trans-activation by expression of the viral protein Tat was demonstrated, significant basal expression was observed. To reduce basal expression, cis-acting negative regulatory elements from the env region of the HIV-1 genome were inserted in the 3' untranslated region of both the luc and DT-A constructs. This dramatically reduced basal expression from the HIV LTR, and now both viral regulatory proteins Tat and Rev were required for maximal trans-activation. Such regulation of DT-A expression might be therapeutically applied to selectively kill HIV-infected cells in acquired immunodeficiency syndrome (AIDS) and AIDS-related complex (ARC).

Gonadotropin-releasing hormone and its receptor in normal and malignant cells.

Gonadotropin-releasing hormone (GnRH) is the hypothalamic factor that mediates reproductive competence. Intermittent GnRH secretion from the hypothalamus acts upon its receptor in the anterior pituitary to regulate the production and release of the gonadotropins, LH and FSH. LH and FSH then stimulate sex steroid hormone synthesis and gametogenesis in the gonads to ensure reproductive competence. The pituitary requires pulsatile stimulation by GnRH to synthesize and release the gonadotropins LH and FSH. Clinically, native GnRH is used in a pump delivery system to create an episodic delivery pattern to restore hormonal defects in patients with hypogonadotropic hypogonadism. Agonists of GnRH are delivered in a continuous mode to turn off reproductive function by inhibiting gonadotropin production, thus lowering sex steroid production, resulting in medical castration. They have been used in endocrine disorders such as precocious puberty, endometriosis and leiomyomata, but are also studied extensively in hormone-dependent malignancies. The detection of GnRH and its receptor in other tissues, including the breast, ovary, endometrium, placenta and prostate suggested that GnRH agonists and antagonists may also have direct actions at peripheral targets. This paper reviews the current data concerning differential control of GnRH and GnRH receptor expression and signaling in the hypothalamic-pituitary axis and extrapituitary tissues. Using these data as a backdrop, we then review the literature about the action of GnRH in cancer cells, the utility of GnRH analogs in various malignancies and then update the research in novel therapies targeted to the GnRH receptor in cancer cells to promote anti-proliferative effects and control of tumor burden.

Long term effects of administration of a gonadotropin-releasing hormone superagonist analog in men with prostatic carcinoma.

Administration of the superagonist analog of GnRH, D-Leu6-GnRH proethylamide, profoundly reduced plasma LH, FSH, testosterone, and dihydrotestosterone levels when given for 6-11 weeks to adult men with prostatic carcinoma. Since patients with prostatic carcinoma can be expected to receive this analog for as long as 3-4 yr, we questioned whether the same degree of reduction could be maintained during chronic administration. In 22 men who had received D-Leu6-GnRH proethylamide for at least 1 yr, LH and testosterone remained at the initial low levels. Plasma dihydrotestosterone concentrations, on the other hand, gradually fell further with long term administration. FSH levels reached a nadir of 5.7 +/- 0.94 (+/- SEM) mIU/ml at 10-11 weeks. Unexpectedly, the plasma levels of this gonadotropin then gradually increased, and between 25 and 97 weeks were approximately 10-15 mIU/ml. This pattern occurred identically in patients receiving either 1 or 10 mg D-Leu6-GnRH proethylamide daily. These data indicate persistent suppression of LH and androgen levels during prolonged therapy and suggest that D-Leu6-GnRH-induced "medical castration" can be maintained with chronic administration.

Use of monoclonal antibody to increase sensitivity and specificity in quantitative immunodiffusion assays.

A mouse monoclonal antibody has been produced which recognizes human liver gamma-cystathionase, Radioiodination of the monoclonal antibody facilitated its use in combination with non-specific precipitating rabbit antisera in classical immunodiffusion assays. The technique may have broad applicability in the detection and quantitation of rare antigens which are difficult to purify but easily recognizable in immunodiffusion assays. It may also be used for the initial detection of monoclonal antibodies to unique antigens of interest.

Familial leukemia and aplastic anemia associated with monosomy 7.

A kindred is described in which eight of 14 patients in one generation had acute nonlymphocytic leukemia or aplastic anemia either alone or terminating in acute nonlymphocytic leukemia. The proband and two siblings in one branch of this kindred presented with aplastic anemia, whereas acute nonlymphocytic leukemia was the presenting feature in the other two branches. Karyotypic evolution from a normal karyotype to monosomy 7 was demonstrated in the proband, and group C monosomy was seen in two other patients. The proband's serum sample inhibited in vitro growth of normal bone marrow colonies. The occurrence of hematologic disease in this kindred appears to be the result of a maternally transmitted trait, and persons younger than 30 years of age appear to have the highest risk of hematologic disease.

Inhibition of HIV production in cells containing an integrated, HIV-regulated diphtheria toxin A chain gene.

Construction of a DNA plasmid that expresses a diphtheria toxin A chain (DT-A) gene under control of human immunodeficiency virus (HIV-1) proteins Tat and Rev has been described. Here the generation of HeLa cell clones containing integrated, HIV-regulated DT-A sequences is reported. Five such clones were identified by their decreased expression of a luciferase reporter gene transiently cotransfected with Tat- and Rev-encoding plasmids. The decreased luciferase expression most probably was due to activation of the integrated DT-A gene because higher luciferase activity could be restored by introducing either DT antitoxin or a gene encoding a mutant, DT-resistant elongation factor 2 (the intracellular target for DT-A). Analysis by polymerase chain reaction (PCR) indicated that all clones expressed DT-A encoding RNA. The clones were then transfected with an HIV proviral clone and were tested for HIV production; all five clones demonstrated substantially impaired HIV production compared with parental HeLa cells, as shown by p24 assays of culture supernatants. Our success in generating these cell lines indicates that extremely low basal expression has been achieved in view of the high cellular lethality of DT-A. HIV-regulated expression of DT-A may be applicable as a gene therapy approach for the acquired immune deficiency syndrome (AIDS), to bring about selective suicide of HIV-infected cells before production of viral progeny.

Production of diphtheria toxin CRM228 in B. subtilis.

The gene coding for a nontoxic diphtheria toxin (DT), tox228, was isolated from lysogenic Corynebacterium diphtheriae and cloned into pBR322. A mature form of the tox228 gene, lacking its signal sequence, was expressed in Bacillus subtilis using a B. amyloliquefaciens alpha-amylase secretion vector. To test the possibility of producing partially deleted DT molecules, which could be used for cell-directed toxin conjugates, a truncated form lacking 151 amino acids from the C-terminus of the DT was generated by oligonucleotide mutagenesis. Both the truncated and intact DT were efficiently secreted into the culture medium. During prolonged cultivation, the truncated form was less stable than the intact DT molecule.

The biology of gonadotropin-releasing hormone and its analogs.

Gonadotropin-releasing hormone (GnRH) is one of the hormones involved in the complex hypothalamic-pituitary-gonadal axis which regulates the release of testosterone from the testes or estrogen from the ovaries. The development of GnRH analogs has helped elucidate the mechanism of action of the natural hormone, and provided possible new ways to treat hormonally related conditions including hormone-dependent cancers, precocious puberty, and endometriosis. The effectiveness of GnRH agonists in clinical use lies in their ability, with long-term administration, to suppress sex-hormone production. GnRH antagonists may eventually replace agonists because they are able to reduce hormone levels without the initial, temporary rise caused by agonists.