Biomedical subjects

E M Rakowicz-Szulczynska

Publications and source records attributed to E M Rakowicz-Szulczynska.

At least 19 recordsLinked to original sources

PubMed2000

Immunohistochemical versus molecular detection of RAK antigens in breast cancer.

RAK antigens p120, p42, and p25 exhibit molecular and immunological similarity to the proteins encoded by HIV-1 and are expressed by 95% of breast and gynecological cancer cases in women and prostate cancer cases in men. Binding of the monoclonal antibody (MAb) RAK-BrI to cancer RAK antigens has been found to be inhibited by a peptide derived from the variable loop V3 of HIV-1. Since MAb RAK-BrI has been developed against denatured froms of breast cancer proteins, and it binds to a short epitope, GRAF, this MAb does not recognize the native, three-dimensional structure of proteins. Subsequently Western blot, after electrophoretic separation in gels with SDS, has been used to detect these unique cancer markers. The current studies were focused on the immunohistochemical evaluation of the novel marker RAK. Serial sections, 5 microm thick, were cut from frozen or Formalin-fixed, paraffin-embedded tissue blocks and immunostained with MAb RAK-BrI. All of the 53 cases of breast cancer tested RAK positive and no differences were observed in the immunohistochemical staining of lobular and ductal carcinoma cases. In contrast, MAb RAK-BrI antigens were detected in only 3 of 15 cases of macroscopically normal breast removed during mastectomy for breast cancer. It is noteworthy that Western blots of breast samples from the same series demonstrated a high expression of three RAK antigens in 20/20 of invasive breast carcinomas, while there was only a very weak expression of RAK antigens in 2/7 of the macroscopically "normal" breast samples. Due to the suspected viral origin of RAK markers, immunohistochemical staining with MAb RAK-BrI might be a useful tool in the early detection of malignant changes occurring in breast tissues.

Antibodies, Monoclonal↗

PubMed2000

Relevance of the viral RAK alpha gene in diagnosis of malignant versus nonmalignant tumors of the ovary and uterus.

Human immunodeficiency virus type 1 (HIV-1)-like antigens RAK (named after the inventor E. M. Rakowicz) p120, p42, and p25, as well as HIV-1-like segments of cancer DNA (RAK gene alpha), have been found before in breast and prostate cancers. The present study focused on determining the value of markers RAK in the diagnosis and prognosis of gynecological cancer. Expression of RAK antigens in ovarian, uterine, cervical, and vulvar cancer, in benign tumors, in tissues adjacent to cancer, and in normal tissues was tested by Western blot hybridization of the electrophoretically separated proteins with monoclonal antibody RAK BrI. The RAK alpha gene was PCR amplified with HIV-1-derived primers SK68 and SK69. RAK antigens p120, p42, and p25 were found in 95% of ovarian, uterine, and cervical cancer cases and in 75% of vulvar cancer cases. The RAK alpha gene was expressed in 100% of cancer cases, in approximately 25% of benign ovarian tumors, and in 40% of benign tumors of the uterus. DNA sequences amplified in all cancer cases exhibited more than 90% homology to HIV-1 gp41 and were encoded for the functional peptide. DNA sequences found in benign tumors contained frameshift mutations and encoded truncated or nonfunctional peptides. Such sequences have not been amplified in normal tissues. RAK antigens and the RAK alpha gene seem to belong to a lentivirus type that is highly related to HIV-1. Beyond the diagnostic value of RAK markers, future cloning of the full viral genome would lead to a better understanding of the etiology of malignant and nonmalignant tumors of reproductive organs and to the development of novel therapeutic approaches.

Antigens, Neoplasm↗

PubMed1999

Giant syncytia and virus-like particles in ovarian carcinoma cells isolated from ascites fluid.

Ovarian cancer cells were isolated from ascites fluid of 30 different patients diagnosed with cystadenocarcinoma of ovaries. Large colonies of malignant ASC cells were observed during the first week of cell growth in vitro. Colony formation was followed by fusion of cells and formation of large multinucleated and highly vacuolated syncytia. In contrast, cells isolated from the ascites fluid produced by patients with benign mucinous cystadenoma of ovaries did not form syncytia. Nonmalignant Brenner tumor cells, isolated from the ascites fluid, also did not form syncytia. Syncytia, but not the nonmalignant tumor cells, were immunofluorescence stained with an anti-human immunodeficiency virus type 1 (HIV-1) gp120 monoclonal antibody (MAb) and MAb RAK-BrI. Both MAbs recognized cancer-associated antigens RAK (for Rakowicz markers) p120, p42, and p25. Exposure of ASC cells to either the anti-HIV-1 gp120 MAb or MAb RAK-BrI inhibited syncytium formation. PCR with HIV-1 Env-derived primers revealed DNA sequences with over 90% homology to HIV-1 gp41 in syncytia and in ovarian cancer cells but not in normal ovary cells. Electron microscopic analysis revealed viral particles, hexagonal in shape (90 nm in diameter), with a dense central core surrounded by an inner translucent capsid and dense outer shell with projections. Negative staining detected membrane-covered particles (100 to 110 nm in diameter) in the cell culture medium. Incubation of normal breast cells with viral particles resulted in drastic morphological changes and syncytium formation by the transformed breast cells. The cytopathic effects of the identified virus resembled those of spumaviruses, which, in addition to their epitopic and genetic homology to HIV-1, might suggest a common phylogeny.

Amino Acid Sequence↗

PubMed1998

Prostate, breast and gynecological cancer markers RAK with homology to HIV-1.

Breast and gynecological cancer-associated antigens RAK p120, p42 and p25 exhibit molecular, immunological and genetic homology to HIV-1 proteins. Normal tissues, including the majority of tissues adjacent to cancer, do not express these unique cancer markers. Antigens RAK are now detected in 100% of prostate cancer and in the majority of prostate benign hyperplasia (BPH) cases. Polymerase chain reaction (PCR) with HIV-1 gp41-derived primers revealed prostate cancer-associated DNA fragments of similar size (140 bp) as in HIV-1 genome. Ninety-five percent of BPH samples obtained from prostate cancer patients tested PCR-positive. For comparison, only 61.9% of BPH samples obtained from cancer-free patients tested PCR-positive. The DNA fragments amplified in prostate cancer and in BPH showed more than 90% homology to the HIV-1 gene for gp41. The obtained results strongly suggest that a retrovirus related to HIV-1 may be associated with cancers of the reproductive system.

Antigens, Neoplasm↗

PubMed1998

Human immunodeficiency virus type 1-like DNA sequences and immunoreactive viral particles with unique association with breast cancer.

RAK antigens p120, p42, and p25 exhibit molecular and immunological similarity to the proteins encoded by human immunodeficiency virus type 1 (HIV-1) and are expressed by 95% of breast and gynecological cancer cases in women and prostate cancer cases in men. The binding of an epitope-specific anti-HIV-1 gp120 monoclonal antibody (MAb) (amino acids 308 to 322) to cancer RAK antigens has been found to be inhibited by a peptide derived from variable loop V3 of HIV-1. Breast cancer DNAs of 40 patients were PCR amplified with HIV-1 gp41-derived primers, and all of the samples were found to be positive. The DNA fragments amplified in seven blindly selected breast cancer samples were sequenced. The breast cancer DNA sequences showed at least 90% homology to the HIV-1 gene for gp41. Antisense oligonucleotides complementary to the HIV-1-like sequences inhibited reverse transcriptase activity and inhibited the growth of breast cancer cells in vitro. Viral particles detected in breast cancer cell lines were strongly immunogold labeled with the anti-HIV-1 gp120 MAb. The results obtained strongly suggest that the long-postulated breast cancer virus may, in fact, be related to HIV-1.

Antibodies, Monoclonal↗

PubMed1997

New protein and PCR markers RAK for diagnosis, prognosis and surgery guidance for breast cancer.

Breast cancer antigens RAK-p120, -p42, -p25 were detected in 100% of breast cancer cases tested (71 cases). Only 10% of adjacent tissue cases tested positive for all three cancer antigens, and 17.5% of the cases tested positive for two antigens only. Eighty-five percent of histologically normal breast tissue samples, isolated either from breast cancer patients or patients with advanced fibrocystic disease, tested RAK-negative, with the exception of low expression of p25, observed in some patients. Polymerase chain reaction (PCR) with HIV-1 gp 41-derived primers revealed cancer-associated DNA fragments of similar size (140 bp) as in HIV-1 genome. Fifty-four percent of cancer adjacent tissues, and 50% of malignancy-free breast tissue samples, tested PCR-negative. It is suggested that genetic predisposition to cancer may be associated with the presence of RAK genes, while expression of RAK antigens marks an already ongoing process of malignant changes.

Biomarkers, Tumor↗

PubMed1996

Immunodetection of squamous cell carcinoma-associated variant of nuclear protein.

A new monoclonal antibody, MAb C63.3, was developed by immunizing mice with the high molecular weight fraction of the cytoplasmic proteins from the cervical squamous cell carcinoma. In Western blotting, MAb C63.3 reacted with cytoplasmic and chromatin antigens expressed by cervical and vulvar squamous cell carcinomas and much weaker with normal cervical tissue and skin cells. Different molecular variants of C63.3 chromatin antigens were found in normal tissues (heavy variants, M(r) 65,000 and 59,000) compared to squamous cell carcinomas (light variant, M(r) 48,000). Diagnostic value of the cancer-associated M(r) 48,000 antigen is discussed. MAb C63.3 was internalized by cells and bound to the chromatin which suggests that antigen(s) C63.3 might represent a receptor for a yet unknown ligand (growth factor). It is suggested that the high molecular weight C63.3 antigens play a role in maintaining the nonmalignant phenotype.

Animals↗

PubMed1996

Inhibition of cancer cell growth by internalized immuno-histone conjugates.

MAb NS 88 directed against breast cancer cells, which is internalized and translocated to the cell nucleus, was conjugated with histone and labeled with 125I. 125I-MAb-histone complexes (M(r) 250,000) were internalized by breast and cervical cancer cells and localized in the cytoplasm and chromatin. Electrophoretic analysis of the cells extracted from the conjugates revealed the same molecular weights of the cytoplasmic and chromatin complexes as those of the native conjugate. Nicotine (0.1%), which suppresses lysosomal degradation, stabilized the conjugates within the cell and prolonged the presence of nondegraded complexes inside the cytoplasm and chromatin from 1 day to at least 3 days. MAb-histone complexes, but not MAb alone, inhibited RNA synthesis and proliferation of cervical and breast cancer cells. A new application of internalized MAbs as the vehicles for protein inhibitors of transcription or replication is discussed.

Antibodies, Monoclonal↗

PubMed1996

PDGF AA as mediator in nicotine-dependent carcinogenesis.

Effect of nicotine on PDGF AA and PDGF BB interaction with cervical cancer SiHa cells was tested. [125I]PDGF AA was internalized by cells and accumulated in the cytoplasm and nucleus (chromatin). In the absence of nicotine, maximal accumulation of [125I]PDGF AA inside the cells occurred after 1 day of incubation, which was followed by a progressive degradation of the growth factor during the next 2, 3 and 5 days of cell exposure. In the presence or 0.001 or 0.01% nicotine, accumulation of [125I]PDGF AA was slightly higher than in the absence of nicotine, and maximal accumulation occurred after 2 days or incubation. In the presence of 0.1 % nicotine, maximal accumulation occurred after 5 days of incubation and was 20 and 14 times higher in the cytoplasm and chromatin, respectively. Nicotine-postponed degradation and increased nuclear accumulation of PDGF AA resulted in activation of RNA synthesis and cell proliferation. PDGF BB, which was not internalized by cells did not respond to nicotine treatment. The proposed mechanism of nicotine-PDGF AA co-carcinogenesis may involve inhibition of growth factor degradation at the lysosomal level and an increased chromatin accumulation of the non-degraded PDGF.

Becaplermin↗

PubMed1994

Growth factor-mediated mechanisms of nicotine-dependent carcinogenesis.

The direct effect of nicotine on the expression of receptors for the tumor necrosis factor alpha (TNF alpha) and transforming growth factor beta (TGF beta) and the internalization, intracellular distribution and stability of these growth factors in cervical cancer cell line SiHa was studied. Nicotine at concentrations in the range 0.05-0.25% inhibited cell growth and it exerted strong apoptotic and cytolytic effects at concentrations above 0.5%. Nicotine at 0.1% stabilized and protected from degradation [125I]TNF alpha and [125I]TGF beta internalized by cervical cancer SiHa cell line. In the absence of nicotine, [125I]TNF alpha and [125I]TGF beta were internalized during the first hour of incubation and localized mainly in the cytoplasm and in smaller amounts in the nucleus. After 1 day of cell exposure to growth factors, only traces of radioactivity were detected inside the cells, which indicated that both growth factors were rapidly degraded. In the presence of nicotine, both [125I]TNF alpha and [125I]TGF beta were detected in high quantities and in a non-degraded form in the cytoplasm and chromatin during 5 days of incubation. In addition to the lack of growth factor degradation, the presence of nicotine induced a nuclear accumulation of growth factors, with up to 37% of the internalized [125I]TGF beta being in the chromatin. An increased intracellular accumulation of [125I]TNF alpha and [125I]TGF beta in cells exposed to nicotine occurred without changes in expression of the cell surface receptors. Nuclear accumulation of TGF beta was followed by increased RNA synthesis and a switch from the growth-promoting action of TGF beta to the strong growth inhibitory effect. Inhibition of the lysosomal degradation of growth factors by nicotine is discussed as a potential mechanism of tobacco-induced carcinogenesis.

Cell Division↗

PubMed1993

Identification of the cell surface and nuclear receptors for NGF in a breast carcinoma cell line.

125I-nerve growth factor (NGF) was found to be internalized and translocated to the nucleus of SKBr5 breast carcinoma cells. The cytoplasm and chromatin isolated from nonmitotic cells accumulated two- and five-fold, respectively, more of 125I-NGF than the cells undergoing mitosis. MAb 20.4 developed against the NGF cell surface receptor immunoprecipitated the 80,000 M(r) receptor from plasma membrane and two protein species from the chromatin; 90,000 M(r) (major band) and 200,000 M(r) (minor band). In SKBr5 cells, binding of NGF to the chromatin did not affect synthesis of rRNA. Proliferation of SKBr5 cells was slightly stimulated by NGF. In control melanoma A875 cells, which express the 230,000 M(r) chromatin receptor, NGF inhibited both rRNA synthesis and cell proliferation. We suggest that the 90,000 M(r) chromatin receptor expressed by SKBr5 cells represents a "nonactive," ligand-binding subunit of the high molecular weight receptor for NGF. The critical role of the chromatin receptor for NGF in rRNA-dependent cell proliferation is discussed.

Breast Neoplasms↗

PubMed1992

Nuclear translocation of monoclonal antibody directed against cell-surface carbohydrate Y determinant.

Monoclonal antibody (MAb) Br 15-6A directed against the carbohydrate Y determinant expressed on tumor cells was found to be internalized and translocated to the nucleus of SK Br 5 breast carcinoma and SW 1116 and SW 707 colorectal carcinoma cells. Intracellular localization of MAb Br 15-6A was determined by cell fraction and by indirect immunofluorescence staining. Internalization of MAb Br 15-6A seems to be mediated by a specific cell surface protein of M(r) 108,000 in colorectal carcinoma cells and M(r) 92,000-96,000 in breast carcinoma cells. The MAb Br 15-6A precipitates an 88,000 M(r) chromatin protein and appears to be bound specifically to two EcoRI-digested and two HincII-chromatin fragments. Another MAb against the Y determinant (MAb Br 55.2) recognizes the same antigens as MAb Br 15-6A, but is not internalized.

Antibodies, Monoclonal↗

PubMed1991

Chromatin and cell surface receptors mediate melanoma cell growth response to nerve growth factor.

Growth response to nerve growth factor (NGF) was tested in the primary melanoma cell line WM 164, which expressed a low level of NGF cell-surface receptor, and in WM 164 cells transfected with cDNA for the cell-surface receptor (TrWM 164 cells), which expressed a higher level of the cell-surface receptor. Neither cell line expressed the chromatin receptor for NGF or internalized NGF. Both cell lines were stimulated to growth by NGF. After 10 d of exposure to NGF, a 230,000 Mr chromatin protein (receptor) was induced in both cell lines; as a result, NGF bound to the chromatin, and ribosomal RNA synthesis and cell proliferation were inhibited. We suggest that the cell-surface and chromatin receptors each mediate a different function of NGF.

Cell Division↗

PubMed1990

Plasma membrane-mediated nuclear uptake and chromatin binding of insulin in tumor cell lines.

Analysis of different cellular fractions after incubation of SW 948 and SW 707 colorectal carcinoma cells or WM 266-4 melanoma cells with 125I-insulin revealed the nondegraded hormone in the chromatin of these cells. Nuclear 125I-insulin was bound to specific fragments of EcoRI-, HaeIII-, and HincII-digested chromatin. A 45-kDa chromatin protein species that binds 125I-insulin was identified. Actinomycin D and cycloheximide inhibited the insulin-stimulated expression of chromatin receptors. Uptake of 125I-insulin by isolated nuclei occurred only in the presence of plasma membranes. Thus, at least some effects of insulin on target cells can be explained by direct gene regulation instead of "second messenger" action.

Cell Membrane↗

PubMed1989

Antagonistic effect of PDGF and NGF on transcription of ribosomal DNA and tumor cell proliferation.

The molecular mechanism by which NGF and PDGF affect growth of tumor cells was tested in human melanoma WM 266-4 and colorectal carcinoma SW 707 cell lines. We present evidence that NGF translocated to the nucleus and bound to the chromatin of SW 707 cells, which express the cell surface and the chromatin receptor for NGF, inhibits ribosomal RNA synthesis which in consequence leads to inhibition of cell proliferation. In WM 266-4 cells, which do not express NGF receptor, NGF does not affect cell proliferation. In contrast, PDGF translocated to the nucleus of both SW 707 and WM 266-4 cells activates ribosomal RNA synthesis. We report here that NGF abolishes PDGF-activated ribosomal RNA synthesis and PDGF-stimulated growth of tumor cells.

Cell Division↗

PubMed1989

Epidermal growth factor (EGF) and monoclonal antibody to cell surface EGF receptor bind to the same chromatin receptor.

Cellular uptake, nuclear translocation, and chromatin binding of epidermal growth factor (EGF) and monoclonal antibodies (MAbs) against the protein domain of the EGF surface receptor (MAb 425) and against the carbohydrate Y determinant on the EGF receptor (MAb Br 15-6A) were analyzed in cell lines that express surface EGF receptor. Both EGF and MAb 425 were translocated to the nucleus and bound in nondegraded form to the chromatin of all cells tested. MAb Br 15-6A was taken up only by SW 948 colorectal carcinoma cells which express EGF receptor whereas neither EGF nor MAb 425 was taken up by SW 707 colorectal carcinoma cells which do not express EGF receptor. MAb 425 immunoprecipitated a 230- to 250-kDa chromatin protein, which appears to be the EGF chromatin receptor. EGF was localized in a single EcoRI DNA fragment suggesting that the chromatin binding was highly specific. Binding of EGF to primarily DNase II-sensitive chromatin regions protected these regions from nuclease action. The role of growth factor binding to chromatin in neoplastic transformation is discussed.

Antibodies, Monoclonal↗

PubMed1989

Rapid and sensitive method to detect expression of growth factor receptors.

A sensitive method to analyze growth factor receptor expression is described that is based on precipitation of the receptor by the growth factor. The precipitate that forms after incubation of pure, membrane-free cytoplasm with the growth factor contains both the receptor and the corresponding mRNA. With this method, nerve growth factor (NGF), epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) receptors were detected in several tumor cell lines thought previously to be receptor-negative. This method is more sensitive than existing cell surface approaches, permitting the detection of growth factor receptor synthesis even in the absence of detectable cell surface expression. The method is comparable in sensitivity to Northern blot hybridization but does not require DNA probes.

Biological Assay↗

PubMed1989

Nuclear uptake of monoclonal antibody to a surface glycoprotein and its effect on transcription.

Nuclear transport and chromatin binding of monoclonal antibody (MAb) ME491, directed against a cell surface glycoprotein, was tested in intact cells and in a cell-free system. After a 24-h incubation with 125I-MAb ME491, the chromatin of melanoma cells and of colorectal carcinoma cells contained approximately 10 and 20%, respectively, of the antibody in nondegraded form. 125I-MAb ME491 was bound to a 55-kDa chromatin protein and localized in two HincII-digested chromatin fragments. Taken up by the nucleus, MAb ME491 inhibited transcription of ribosomal RNA genes by 70%. Nuclear uptake of 125I-MAb ME491 was increased up to ninefold when cells were preincubated with puromycin or actinomycin D. Nuclear uptake of MAb ME491 in a cell-free system was inhibited by ME491 antigen newly synthesized in the cytoplasm. Binding of 125I-MAb ME491 to the newly synthesized ME491 antigen caused precipitation of polysome-bound ME491 mRNA. The effect of MAb ME491 on transcription is discussed.

Antibodies, Monoclonal↗