Clinical experience with non-invasive prenatal screening for single-gene disorders.

OBJECTIVE: To assess the performance of a non-invasive prenatal screening test (NIPT) for a panel of dominant single-gene disorders (SGD) with a combined population incidence of 1 in 600. METHODS: Cell-free fetal DNA isolated from maternal plasma samples accessioned from 14 April 2017 to 27 November 2019 was analyzed by next-generation sequencing, targeting 30 genes, to look for pathogenic or likely pathogenic variants implicated in 25 dominant conditions. The conditions included Noonan spectrum disorders, skeletal disorders, craniosynostosis syndromes, Cornelia de Lange syndrome, Alagille syndrome, tuberous sclerosis, epileptic encephalopathy, SYNGAP1-related intellectual disability, CHARGE syndrome, Sotos syndrome and Rett syndrome. NIPT-SGD was made available as a clinical service to women with a singleton pregnancy at ≥ 9 weeks' gestation, with testing on maternal and paternal genomic DNA to assist in interpretation. A minimum of 4.5% fetal fraction was required for test interpretation. Variants identified in the mother were deemed inconclusive with respect to fetal carrier status. Confirmatory prenatal or postnatal diagnostic testing was recommended for all screen-positive patients and follow-up information was requested. The screen-positive rates with respect to the clinical indication for testing were evaluated. RESULTS: A NIPT-SGD result was available for 2208 women, of which 125 (5.7%) were positive. Elevated test-positive rates were observed for referrals with a family history of a disorder on the panel (20/132 (15.2%)) or a primary indication of fetal long-bone abnormality (60/178 (33.7%)), fetal craniofacial abnormality (6/21 (28.6%)), fetal lymphatic abnormality (20/150 (13.3%)) or major fetal cardiac defect (4/31 (12.9%)). For paternal age ≥ 40 years as a sole risk factor, the test-positive rate was 2/912 (0.2%). Of the 125 positive cases, follow-up information was available for 67 (53.6%), with none classified as false-positive. No false-negative cases were identified. CONCLUSIONS: NIPT can assist in the early detection of a set of SGD, particularly when either abnormal ultrasound findings or a family history is present. Additional clinical studies are needed to evaluate the optimal design of the gene panel, define target populations and assess patient acceptability. NIPT-SGD offers a safe and early prenatal screening option. © 2021 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of International Society of Ultrasound in Obstetrics and Gynecology.

Circulating tumor DNA in neoadjuvant-treated breast cancer reflects response and survival.

BACKGROUND: Pathologic complete response (pCR) to neoadjuvant chemotherapy (NAC) is strongly associated with favorable outcome. We examined the utility of serial circulating tumor DNA (ctDNA) testing for predicting pCR and risk of metastatic recurrence. PATIENTS AND METHODS: Cell-free DNA (cfDNA) was isolated from 291 plasma samples of 84 high-risk early breast cancer patients treated in the neoadjuvant I-SPY 2 TRIAL with standard NAC alone or combined with MK-2206 (AKT inhibitor) treatment. Blood was collected at pretreatment (T0), 3 weeks after initiation of paclitaxel (T1), between paclitaxel and anthracycline regimens (T2), or prior to surgery (T3). A personalized ctDNA test was designed to detect up to 16 patient-specific mutations (from whole-exome sequencing of pretreatment tumor) in cfDNA by ultra-deep sequencing. The median follow-up time for survival analysis was 4.8 years. RESULTS: At T0, 61 of 84 (73%) patients were ctDNA positive, which decreased over time (T1: 35%; T2: 14%; and T3: 9%). Patients who remained ctDNA positive at T1 were significantly more likely to have residual disease after NAC (83% non-pCR) compared with those who cleared ctDNA (52% non-pCR; odds ratio 4.33, P = 0.012). After NAC, all patients who achieved pCR were ctDNA negative (n = 17, 100%). For those who did not achieve pCR (n = 43), ctDNA-positive patients (14%) had a significantly increased risk of metastatic recurrence [hazard ratio (HR) 10.4; 95% confidence interval (CI) 2.3-46.6]; interestingly, patients who did not achieve pCR but were ctDNA negative (86%) had excellent outcome, similar to those who achieved pCR (HR 1.4; 95% CI 0.15-13.5). CONCLUSIONS: Lack of ctDNA clearance was a significant predictor of poor response and metastatic recurrence, while clearance was associated with improved survival even in patients who did not achieve pCR. Personalized monitoring of ctDNA during NAC of high-risk early breast cancer may aid in real-time assessment of treatment response and help fine-tune pCR as a surrogate endpoint of survival.

Primary closure or rhomboid excision and Limberg flap for the management of primary sacrococcygeal pilonidal disease? A meta-analysis of randomized controlled trials.

AIM: Sacrococcygeal pilonidal disease is a common condition afflicting the young male working and student population, resulting in considerable pain, embarrassment and loss of work days. Controversy surrounds the most appropriate surgical approach to achieve low recurrence rates whilst minimizing morbidity and permitting an early return to work. This study aims to review the published literature comparing excision followed by either primary suture or rhomboid flap repair. METHODS: PubMed, EMBASE, MEDLINE and The Cochrane Library were systematically reviewed, by two independent investigators, for relevant randomized controlled trials. Keywords and MeSH terms included 'pilonidal disease', 'primary suture/repair', 'rhomboid flap' and 'limberg/modified Limberg flap'. 'Related study' function and manuscript bibliographies were searched for further relevant studies. Study quality was assessed using the Jadad score. Meta-analysis was performed on pooled data, utilizing a random effects model when heterogeneity was high and a fixed effects model when heterogeneity was low. The primary end-point assessed was disease recurrence. Secondary end-points included wound dehiscence, pain scores, hospital stay and return to work. RESULTS: Six studies were eventually included for pooled analysis following exclusion of randomized controlled trials with poor methodology. Two studies compared 'off-midline' (Karydakis) primary suture with the Limberg flap repair. Six hundred and forty-one patients were included (331 flap repairs). Rhomboid flap excision demonstrated a trend towards less disease recurrence (P = 0.07), lower wound infection (P = 0.001) and dehiscence (P = 0.01). However, no significant difference was found for pain scores, hospital stay or return to work. CONCLUSION: The current published literature supports the use of the rhomboid flap excision and the Limberg flap-repair procedures over primary midline suture techniques for the elective management of primary pilonidal disease. Further high-quality studies are necessary to compare flap with off-midline repairs.

Utility of incorporating a gene-based lung cancer risk test on uptake and adherence in a community-based lung cancer screening pilot study.

Based on the results of randomized control trials, screening for lung cancer using computed tomography (CT) is now widely recommended. However, adherence to screening remains an issue outside the clinical trial setting. This study examines the utility of biomarker-based risk assessment on uptake and subsequent adherence in a community screening study. In a single arm pilot study, current or former smokers > 50 years old with 20 + pack year history were recruited following local advertising. One hundred and fifty seven participants volunteered to participate in the study that offered an optional gene-based lung cancer risk assessment followed by low-dose CT according to a standardised screening protocol. All 157 volunteers who attended visit 1 underwent the gene-based risk assessment comprising of a clinical questionnaire and buccal swab. Of this group, 154 subsequently attended for CT screening (98%) and were followed prospectively for a median of 2.7 years. A participant's adherence to screening was influenced by their baseline lung cancer risk category, with overall adherence in those with a positive scan being significantly greater in the "very high" risk group compared to "moderate" and "high" risk categories (71% vs 52%, Odds ratio = 2.27, 95% confidence interval of 1.02-5.05, P = 0.047). Those in the "moderate" risk group were not different to those in the "high" risk group (52% and 52%, P > 0.05). In this proof-of-concept study, personalised gene-based lung cancer risk assessment was well accepted, associated with a 98% uptake for screening and increased adherence for those in the highest risk group.

Functional characteristics of Peyer's patch lymphoid cells. II. Lipopolysaccharide is thymus dependent.

This study shows that LPS is not mitogenic in cultures containing B cells, or B cells and accessory adherent cells or ME, unless T cells are present. This observation rules out models of induction of antibody synthesis in which it is assumed that the delivery of a mitogenic signal by the interaction of LPS with the membrane of the B cell is in itself sufficient for B-cell induction (19). Further, it makes unlikely the proposed extrapolation of such a model to other so-called thymus-independent antigens, e.g., PVP, levan, dextran, and SIII (19). The mitogenic action of LPS appears to be due to its ability to complete an inductive stimulus to B cells (13). We interpret the observed thymus dependence of the B-cell response to LPS in light of a model in which two signals are obligatory for B-cell induction (14). The first signal in the inductive pathway is delivered to the antigen-sensitive cell via a conformational change in the receptor upon interaction with antigen. The second signal is delivered via the thymus-derived cooperating system. Since LPS can induce immune responses to both immunogenic and nonimmunogenic ligands (9-13) we envision that one signal is delivered to the B cell via specific binding of the ligand to the B-cell antigen receptor, while a second signal is delivered as a result of T-cell cooperation via membrane-bound LPS. This has been termed abnormal induction (20). In this example LPS is the foreign membrane-bound determinant in question although histocompatibility antigens (21, 22), viral determinants, or surface bound lectins could act similarly. In light of the above model, one observation should be pointed out. LPS inhibits the induction of a SRBC response in normal Peyer's patch cells to which adherent cells or ME is added. This inhibition appears to be a T-cell-mediated effect because it is abolished by partial depletion of the T-cell population by antitheta treatment. Since the induction of IgM producing PFC is being measured, the T-cell-dependent LPS inhibition could act either (a) by induction of T-cell "suppression" (23, 24) of the normal cooperating system required for a SRBC response, or (b) by the induction of such high levels of cooperating function (13) as to be inhibitory to a SRBC IgM response. Our observations contrast sharply with prior reports which describe LPS as a thymus-independent antigen (2-4) and a B-cell mitogen (5-8) capable of stimulating immune responses in the absence of T-cell cooperation (2-12). This demonstration of the thymus dependence of LPS stimulation has been possible because Peyer's patches from congenitally athymic (nude) mice are functionally a highly purified B-cell population devoid of T cells and accessory adherent cells. In this respect, earlier studies relied on nude spleen cultures and spleen cultures from thymectomized, lethally irradiated, and bone marrow-reconstituted mice (3, 4, 6-13). These spleen cultures which contain B cells and accessory adherent cells are recognized to be deficient but not devoid of the thymus-derived contribution to the inductive stimulus (12, 13). It could be argued that the presence of T cells and adherent cells is in fact required for the antigen-specific effect and not for the LPS effect. However, this is unlikely since our experiments show that LPS is not directly mitogenic for B cells and does not stimulate background anti-SRBC PFC. It seems unlikely that Peyer's patch antigen-sensitive cells differ from antigen-sensitive cells in the spleen in their mechanism of induction. We have shown that Peyer's patch B cells can be specifically induced by antigen, and Peyer's patch T cells mediate cooperating and killer functions. Alternately, the possibility that Peyer's patch B cells were not stimulated by LPS as a result of prior cryptic exposure to LPS (13) in the intestinal tract was excluded since cultures containing B cells, T cells, and adherent cells or ME were stimulated to DNA synthesis by LPS. The reason that certain antigens appear to be thymus independent may be that their repeating polymeric nature permits inductive interactions at very low levels of thymus-derived cooperation (see reference 20 for quantitative considerations). It has been stated that the inductive properties of all thymus-independent antigens are directly related to their ability to act as B-cell mitogens (19). The observation that LPS is thymus dependent for its B-cell mitogenic activity makes us question the thymus independence of any antigen.

Genetic control of cytolytic T-lymphocyte responses. I. Ir gene control of the specificity of cytolytic T-lymphocyte responses to trinitrophenyl-modified syngeneic cells.

The ability of cytotoxic T lymphocytes (CTL) induced in vitro to trinitrophenyl (TNP)-modified syngeneic cells to cross-reactively lyse a TNP allogeneic spleen target varies among inbred mouse strains. The cross-reactive CTL phenotype was found to be histocompatibility 2 (H-2) linked and to be dominant in F1 hybrid mice. All strains investigated demonstrated cross-reactivity except for some strains bearing portions of the H-2k haplotype. The gene(s) controlling this response maps to the K and/or I-A region of the H-2 complex. We have termed the immune response (Ir) gene responsible for controlling the specificity of CTL induced to TNP-modified syngeneic cells Ir-X-TNP.

Genetic control of cytolytic t-lymphocyte responses. II. The role of the host genotype in parental leads to F1 radiation chimeras in the control of the specificity of cytolytic T-lymphocyte responses to trinitrophenyl-modified syngeneic cells.

Bone marrow cells from C3H (H-2k) mice, a strain that does not exhibit cross-reactive lysis of trinitrophenyl (TNP)-modified allogeneic targets, were allowed to mature in heavily irradiated (B6 times C3H)F1 (H-2b/k) recipients, an F1 hybrid that does demonstrate cross-reactive lysis. Spleen cells from these chimeric mice were removed after 3-4 mo and by H-2 typing shown to be of C3H origin. These cells were found to be tolerant to B6 alloantigens by mixed lymphocyte reaction and cell-mediated cytotoxicity and, when stimulated in vitro with TNP-modified syngeneic cells, now cross-reactively lysed TNP-modified allogeneic targets. These studies demonstrate that the host environment where T cells differentiate influences the specificity of the primary cytolytic T-lymphocyte (CTL) response to TNP-modified syngeneic antigens.

Cytotoxic T lymphocytes specific for I region determinants do not require interactions with H-2K or D gene products.

Gene products coded for by the major hisocompatibility complex (MHC) can serve as target antigens for cytotoxic T lymphocytes (CTL) (1). A variety of test systems are available which have yielded information consistently reinforcing the importance of this complex of genes in the generation and effector phases of the cytotoxic immune response. Originally, it was shown that allogeneically-induced CTL had specificity primarily for the products of the K and D loci of the mouse H-2 complex (2). More recently this has also been found to be the case for xenogeneic immunizations (3,4). Additional examples of T cell-mediated lysis have been reported involving viral-infected or chemically- modified syngeneic stimulating and target cells in which homology at H-2K or H-2D was required between the responding and target cells for appreciable lysis to occur (5-7). Moreover, CTL specific for minor histocompatability antigens are able to lyse only target cells bearing these membrane antigens and sharing a common H-2K or H2-D gene product with the effector (8,9). Two hypotheses have been proposed to explain the requirement for H-2 identity between effector and targets in these systems. CTL may recognize new antigenic determinants created by the interaction of the modifier with syngeneic K and D gene products. Alternately, a dual recognition system my exist, requiring an antigen-specific receptor as well as a second receptor with specificity for homologous H-2K or H-2D determinants (5). Neither model can be excluded at this time. The I region also contains genes coding for histocompatibility loci since animals differing at the I-A or I-C regions of the H-2 complex reject skin grafts (10-12), though less rapidly than mice differing at the H-2K or H-2D regions, Also CTL can be generated to I region determinants but less efficiently than CTL specific for H-2K or H-2D gene products (12-14). The question can therefore be raised, whether the I region minor histocompatibility loci function independently from the H-2K or H-2D loci or whether I region-specific cytolysis requires the participation of H-2K or H-2D gene products of the target cell. This communication illustrates the generation of CTL showing specificity for I region determinants in primary mixed lymphocyte cultures. Further, we demonstrate by genetic analysis and byt eh use of speficit alloantisera that CTL directed to Ia determinants (a) do not see these antigens as modifications of H-2K or H-2D gene products but as independent gene products coded for by the I region, and (b) they do not require interaction with target cells bearing the same H-2K or H-2D gene product as the effect CTL.

Cytotoxic T lymphocytes induced against allogeneic I-region determinants react with Ia molecules on trinitrophenyl-conjugated syngeneic target cells.

The major histocompatibility complex codes for determinants which are recognized by and serve as targets for cytolytic T lymphocytes (CTL) (1). Antigens coded for by the K and D loci of the H-2 complex can activate xenogeneic or allogeneic CTL (2,3). In addition, the H-2K or H-2D gene products function as those molecules against which syngeneic CTL responses specific for chemical, viral, and minor H antigens are directed (4-8). It has recently been shown that Ia determinants can also serve as target antigens for distinct but weaker CTL responses (9-13). Those clones which recognize Ia antigens see them independently of K- or D- coded antigens as shown in genetic studies and by antisera-blocking experiments (12,13). We have proposed that the existence of clones of CTL specific for I-region-coded determinants is not fortuitous; rather these clones specifically recognize Ia determinants and may have an immunoregulatory role. These CTL may affect those immune functions which are at least partially dependent on or controlled by I-region-coded molecules. Two predictions can be made and tested concerning the role of Ia determinants in cytolytic systems and the role, if any, of I-region- specific CTL in regulating the immune response: (a) that if as we and others have shown, certain Ia specificities can serve as a third series of major histocompatibility antigens, then Ia antigens should be susceptible to the same types of antigenic modifications as H-2K- or H-2D-coded structures and thus serve as targets for CTL directed against modified-self in selected systems; and (b) that allogeneically induced I-region-specific CTL should demonstrate cross-reactivity with targets bearing modified syngeneic I-region-coded determinants. Data will be present which demonstrates that trinitrophenyl (TNP)-modified syngeneic I-region determinants can serve as targets for CTL induced by allogeneic Ia antigens.

Clustering of cytokine genes on mouse chromosome 11.

The presence of positionally conserved amino acid residues suggests that the mouse proteins TCA3, P500, MIP1-alpha, MIP1-beta, and JE are members of a single gene family. These proteins are activation specific and can be expressed by both myeloid and lymphoid cells. MIP1-alpha/MIP1-beta and MCAF (the putative human homologue of JE) act as chemotactic and activating agents for neutrophils and macrophages, respectively. The functions of TCA3 and P500 are unknown. We have used interspecies somatic cell hybrids and recombinant inbred mouse strains to show that the genes encoding TCA3, MIP1-alpha, MIP1-beta, and JE (provisionally termed Tca3, Mip-1a, Mip-1b, and Sigje, respectively) map as a cluster on the distal portion of mouse chromosome 11 near the Hox-2 gene complex. DNA sequence analysis indicates that the P500 and TCA3 proteins are encoded by alternative splicing products of one genomic gene. Additionally, the genes encoding TCA3 and JE are found to be strikingly similar with respect to the positions of intron-exon boundaries. Together, these data support the model that the cytokines TCA3, P500, MIP1-alpha, MIP1-beta, and JE are encoded by a single cluster of related genes. The gene encoding IL-5 (Il-5), which acts as a T cell-replacing factor, a B cell growth factor, and an eosinophil differentiation factor, is also mapped to mouse chromosome 11.Il-5 maps approximately 25 cM proximal to the Tca-3 gene and appears tightly linked to a previously described gene cluster that includes Il-3, Il-4, and Csfgm. We discuss the potential relevance of the two cytokine gene clusters described here with particular attention to specific human hematologic malignancies associated with chromosomal aberrations at corresponding locations on human chromosomes 5 and 17.

Genetic testing in the workplace: a view from the USA.

Progress in human genetics, accelerated by the Human Genome Project, is leading to a rapid proliferation in the number of genetic tests. Although they have their benefits, genetic tests have in the past been used by various societal institutions, including employers, to discriminate against and stigmatize individuals. In the light of the increase in the number of tests and some current reports of discrimination in employment, a variety of legal and regulatory measures have been proposed to prevent genetic discrimination in the workplace.

Inhibition of oral carcinogenesis by a protease inhibitor.

The effect of the Bowman-Birk inhibitor (BBI) and soybean trypsin inhibitor (SBTI) on experimental 7,12-dimethylbenz[a]anthracene [(DMBA) CAS: 57-97-6]-induced oral carcinogenesis in Syrian male hamsters was examined. All treatments were applied topically on both cheek pouches for 20 weeks, and the animals were then sacrificed. Gross and microscopic evaluations revealed a statistically significant reduction in the number of invasive carcinomas, the total number of tumors, and the tumor mass for the DMBA + BBI treatment group compared to animals treated with DMBA alone, DMBA and autoclaved BBI (a preparation in which protease inhibitor activity is destroyed), or DMBA + SBTI. A protease activity (with the use of Boc-Val-Pro-Arg-MCA as substrate) was measured and found to be elevated about tenfold in tumorous and nontumorous tissue from DMBA-treated cheek pouches. This protease activity was found to be decreased in the DMBA and BBI treatment group but not in the DMBA + SBTI or DMBA and autoclaved BBI treatment groups, as compared to the protease activity in the DMBA treatment group. Partial characterization of the Boc-Val-Pro-Arg-MCA hydrolyzing activity with diisopropyl fluorophosphate suggests that the proteolytic activity is a serine protease. Iodoacetamide and diethyl pyrocarbonate also inhibit enzyme activity, suggesting that other residues may be necessary for catalysis, possibly including cysteine and histidine. Our results suggest that this protease activity may play a role in DMBA-induced cheek pouch carcinogenesis.

Effects of praziquantel, a new antischistosomal drug, on the mutation and transformation of mammalian cells.

Praziquantel (PQ), a tetrahydroquinoline derivative, is a new and clinically effective antischistosomal drug, which has been shown to lack or to possess very weak mutagenic activity. However, in bacteria, this compound can act as a weak comutagen that increases the mutagenicity of several chemical mutagens and carcinogens. We have found that PQ can act as a very weak comutagen in animal cells. At 10 to 50 micrograms/ml, PQ increased the mutagenicity of N-methyl-N'-nitro-N-nitrosoguanidine and 2-methoxy-6-chloro-9-[3-(2-chloroethyl)amino-propylamino]acridine dihydrochloride about 2-fold in Chinese hamster V-79 cells. In C3H/10T 1/2 mouse embryo cells, PQ exhibited only negligible comutagenic activity. PQ did not oncogenically transform C3H/10T 1/2 cells but had a pronounced effect on 3-methylcholanthrene-induced transformation of these cells. When PQ was coadministered with or added after 3-methylcholanthrene treatment, the number of type III foci produced was about 5-fold lower than in cultures treated with 3-methylcholanthrene alone. Therefore, PQ can inhibit type III focus formation in C3H/10T 1/2 cells.

Mutation of Chinese Hamster V79 cells and transformation and mutation of mouse fibroblast C3H/10T1/2 clone 8 cells by aflatoxin B1 and four other furocoumarins isolated from two Nigerian medicinal plants.

Mutation by aflatoxin B1 (AFB1), imperatorin, marmesin, chalepin, and 8-methoxypsoralen (MOP), with and without black light (BL; long-wavelength ultraviolet light) activation, was determined at the hypoxanthine-guanine phosphoribosyltransferase locus (8-azaguanine resistance) in Chinese hamster V79 cells and at the ouabain locus in mouse C3H/1OT1/2 cells. Transformation by these furocoumarins under the same activation conditions was also investigated in C3H/1OT1/2 cells. In V79 cells, AFB1 induced a 4-fold maximum mutation frequency over controls under BL activation at a concentration of 5 micrograms/ml; marmesin induced a 2-fold increased mutation frequency at 1.5 micrograms/ml; MOP induced a 19-fold increase at 10 micrograms/ml; chalepin induced a 3-fold increase at 5 micrograms/ml; and imperatorin induced a 20-fold increase at 10 micrograms/ml. Essentially no mutation was observed at the ouabain-resistant (Ouar) locus in C3H/1OT1/2 cells with any of these compounds. In the transformation assays, type II and type III foci were observed at a 1-microgram/ml addition of AFB1 with or without BL activation; while with MOP and imperatorin, these types of foci were observed only with BL activation. Marmesin, although relatively more cytotoxic than the other furocoumarins studied, with a 50% lethal dose of less than 0.5 micrograms/ml, was not as mutagenic or potentially carcinogenic as were AFB1, imperatorin, or MOP with BL activation. These furocoumarins are considered to be involved in the etiology of the high incidence of skin cancer in Nigeria. Our experiments reinforce that concept and suggest that exposure to these furocoumarins may constitute a real carcinogenic hazard.

Influence of benzoflavone on aflatoxin B1-induced cytotoxicity, mutation, and transformation of C3H/10T1/2 cells.

Aflatoxin B1 (AFLB1), a metabolite of the fungus Aspergillus flavus, is hepatotoxic and hepatocarcinogenic in several animal species and is thought to play an etiological role in human liver cancer. C3H/10T1/2 clone 8 mouse embryo fibroblasts are killed, mutated, and morphologically transformed byAFLB1. 7,8-Benzoflavone, a known inhibitor of aryl hydrocarbon hydroxylase, inhibits this enzymatic activity in C3H/10T1/2 cells. Furthermore, benzoflavone inhibits the binding of AFLB1, to the DNA of C3H/10T1/2 cells. Benzoflavone also inhibits AFLB1-induced cytotoxicity and mutation of C3H/10T1/2 cells, as well as inhibiting the activation of AFLB1 into mutagenic metabolites capable of reverting the Ames Salmonella tester strain TA98. Interestingly, benzoflavone had no effect on the oncogenic transformation of these cells by AFLB1. Therefore, benzoflavone inhibits the DNA binding, cytotoxic, and mutagenic effects of AFLB1 but does not reduce the morphological transformation of C3H/10T1/2 cells by this mycotoxin.

Potential intracellular target proteins of the anticarcinogenic Bowman Birk protease inhibitor identified by affinity chromatography.

The soybean-derived Bowman Birk inhibitor (BBI) has been shown to inhibit carcinogenesis in both in vitro and in vivo model systems. In the present study, we have utilized a BBI affinity column to determine whether cellular enzymes, present in C3H/10T1/2 cells, specifically interact with this inhibitor. Using this technique, we have identified three proteins with masses of about 70, 60, and 50 kilodaltons. Cell fractionation experiments demonstrate that the 60- and 50-kilodalton proteins are present in the 10,000 x g pellet (lysosomal/golgi fraction) of C3H/10T1/2 cell homogenates. We have also identified two proteins with masses of 60 and 50 kilodaltons which bind to the BBI affinity column in fibroblasts from patients having Bloom syndrome. BBI as well as several other protease inhibitors has been shown previously to reduce the frequency of spontaneous chromosomal aberrations in these cells. Our results indicate that the 50- and 60-kilodalton proteins we have identified by affinity chromatography are present in both mouse and human cells and further suggest that these proteins are potential intracellular targets of the BBI in these cells.

Human fibroblasts contain a proteolytic activity which is inhibited by the Bowman-Birk protease inhibitor.

The Bowman Birk protease inhibitor (BBI) has been shown to be an effective suppressor of carcinogenesis in vivo and in vitro. In this report we demonstrate that normal human fibroblasts and Bloom cells contain a BBI-inhibitable proteolytic activity. The enzyme cleaves gelatin, has a molecular mass of 43 kDa, and is located in the cytosol. This activity has maximal activity at pH 8 and was inhibited by diisopropylfluorophosphate but was not affected by EDTA or 1,10-phenanthroline, indicating that this enzyme is a serine protease. We have reported previously that a similar BBI-inhibitable activity is present in C3H/10T1/2 mouse embryo fibroblast cells. Our results suggest that a common "target enzyme" of the BBI is present in mouse and human cells.

Enhanced expression of c-myc and decreased expression of c-fos protooncogenes in chemically and radiation-transformed C3H/10T1/2 Cl 8 mouse embryo cell lines.

c-abl, c-fos, c-Ha-ras, c-myc, and c-mos were expressed whereas c-sis, c-fms, c-rel, c-src, and c-myb expression was not detectable in C3H/10T1/2 Cl 8 (10T1/2) cells and in eight chemically and radiation-transformed 10T1/2 cell lines. The expression of c-abl, c-fos, c-Ha-ras, and c-myc was growth-related in nontransformed 10T1/2 cells. c-abl and c-fos expression increased at confluence by 5- and 9-fold, respectively, compared to that in log phase cells. c-Ha-ras and c-myc transcripts were most abundant in log phase cells and decreased by 70 and 50%, respectively, in confluent cells. There were no significant growth-related changes in the expression of c-Ha-ras, c-myc, or c-abl in methylcholanthrene-transformed Cl 15 cells. The c-fos transcript was not detected in Cl 15 cell cultures. c-abl, c-fos, c-ras, and c-myc were expressed in whole C3H mouse embryo tissue, mouse liver, and 10T1/2 cells. Sizes of these protooncogene transcripts in 10T1/2 cells were the same as those in whole embryo tissue, except that 10T1/2 cells did not express the 8.2-kilobase abl transcript. At subconfluence, equivalent low levels of c-mos expression were observed in nontransformed and in the eight transformed 10T1/2 cell lines. The level of c-abl expression was similar in the nontransformed and in the eight transformed cell lines, but there was a new 8.2-kilobase transcript in the transformed MCA Cl 15 cell line. c-fos was expressed in 10T1/2 cells but was not detectable or greatly reduced in eight transformed cell lines. c-Ha-ras was expressed to a similar extent in eight transformed cell lines and in nontransformed 10T1/2 cells. In the UVC-4 transformed cell line, extra 3.3-kilobase Ha-ras and 7.5-kilobase Ki-ras transcripts were observed. c-myc was expressed at 4- to 7-fold higher levels in six transformed cell lines compared to 10T1/2 cells. There were no major rearrangements in or amplification of the c-myc gene in three transformed cells overexpressing this gene 5-fold. These studies show that enhanced expression of c-myc and decreased expression of c-fos correlate with the chemically and radiation transformed states of 10T1/2 cells. Changes in c-fos and c-myc oncogene expression may be casually linked to late stages of neoplastic transformation in these chemically and radiation transformed 10T1/2 cell lines.