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Seven breakdown products of tRNA were quantitated by high pressure liquid chromatography in urine and were related to the creatinine content. In the urine of 26 of 27 patients with 13 different malignancies, there was an elevation of one or more of these "markers." The levels of excretion vary approximately with the stage of the disease.
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Ten aminoacyl transfer RNA's prepared from human malignant trophoblastic cells (BeWo line) were compared with the corresponding aminoacyl transfer RNA's from normal human chorionic tissue by cochromatography on a RPC-5 column. Phenylalanyl transfer RNA (Phe-tRNA) of BeWo cells had, in addition to the single species of Phe-tRNA found in normal chorionic tissues, an early eluting component. When Phe-tRNA from the chorion was exposed to mild acid, which selectively excises the Y base, it eluted in the same position as the early eluting Phe-tRNA of BeWo cells. Therefore, the BeWo Phe-tRNA is partially undermodified. Tyrosyl transfer RNA of BeWo cells exhibited a broad-based peak which eluted later than the normal and probably consists of two or more tyrosyl transfer RNA's. Seryl transfer RNA of BeWo cells showed two peaks of acceptor activity, while seryl transfer RNA of normal chorion had a third peak that eluted at a higher salt concentration. In addition, in an early eluting methionyl and lysyl transfer RNA and in a late eluting arginyl transfer RNA from BeWo cells and normal charion, quantitative alterations were detected. The remaining four transfer RNA's, leucyl, aspartyl, valyl, and histidyl, from the two sources did not show any significant differences in elution profiles. These alterations of the chromatographic profile appeared to be due to new or altered species of transfer RNA. They were not due to differences in the aminoacyl transfer RNA synthetase. The transfer RNA methyltransferase capacity of the enzymes from BeWo cells was 2-fold higher than that of the enzymes extracted from the chorion.
The special feature of nucleolar morphology which reflects the intensity of ribosomal RNA precursor molecule formation and thus also the intensity of proteosynthesis was studied in contact surface preparations from tissue samples of 28 patients with epithelial tumors of the bladder and in tissue samples of bladder mucosa from 16 patients with prostata hypertrophy (7 of them suffered also from cystolithiasis). In malignant lesions of the bladder mucosa, especially in tumors of medium and high grade malignancy, very low values of metabolically resting cells with ring shaped nucleoli and micronucleoli were found.
In most cervical carcinoma cells the E6 and E7 genes of specific human papillomaviruses are transcribed from viral sequences integrated into host cell chromosomes. Glucocorticoids activate the promoter elements of various human papillomaviruses in transient-expression assays. We have analyzed the effect of dexamethasone on the transcription rate of human papillomavirus 18 E6 and E7 genes integrated at different chromosomal sites in four cervical cancer cell lines. Dexamethasone led to an increase in the transcription rate of the integrated E6-E7 sequences in C4-1 and C4-2 cells but led to a decrease in SW 756 cells and did not affect the transcription rate in HeLa cells. However, when the viral promoter elements derived from HeLa or SW 756 cells, in which dexamethasone does not activate transcription of the integrated E6-E7 sequences, were tested in transient-expression assays within the same cell lines, dexamethasone consistently activated the viral promoter. It thus appears that dominant regulatory mechanisms presumably depending on the chromosomal integration site are able to override the response of the viral promoter to steroid hormones. The growth rate of all dexamethasone-treated cell lines correlated consistently with the expression of the papillomavirus E6 and E7 genes, supporting their role in the maintenance of the proliferative phenotype of cervical carcinoma cells. Since human papillomaviruses are integrated into the host cell genome at variable, presumably randomly selected chromosomal loci, regulatory mechanisms that influence viral gene expression, and hence cell growth, may differ among cancers of independent clonal origin.
Human papillomavirus (HPV) is associated with specific benign and malignant lesions of the epithelial and mucosal surfaces. Of the sexually transmitted types, HPV type 16 (HPV-16) is the most commonly associated with carcinoma of the uterine cervix. Expression of the E6/E7 open reading frame of the viral genome is considered critical in the development of neoplasia. Using the CaSki cervical carcinoma cell line as a model system, we have adapted the polymerase chain reaction to quantify the transcripts expressed from this region. It was found that 97.1% of the total spliced transcript is E6*I, which putatively encodes the E7 oncoprotein, while E6*II comprises 2.9% of spliced product. The ratio of E6*I to E6*II expression may be an important parameter in evaluating the disease risk associated with HPV-16 infection.
BACKGROUND: Localization of human regenerating gene (reg) mRNA and its product was investigated in normal and neoplastic human pancreas with the in situ hybridization method and immunohistochemical studies. METHODS: Both reg mRNA and reg protein were observed in acinar cells of the pancreas, but neither was found in ductal or islet cells. Immunoreactive reg was observed in an acinar cell carcinoma, a pancreatoblastoma, a solid and cystic tumor, and 5 of 20 duct cell carcinomas, but it was not found in 15 endocrine tumors and 2 microcystic adenomas. RESULTS: For comparison with reg, alpha-1-antitrypsin (AAT), lysozyme, chromogranin A (CMG), CA 19-9, carcinoembryonic antigen (CEA), cytokeratin, vimentin, and alpha-fetoprotein (AFP) were assessed in those tumors. An acinar cell carcinoma and a pancreatoblastoma had positive results for AAT, lysozyme, cytokeratin, and AFP but negative results for vimentin. An acinar cell carcinoma showed cells focally immunoreactive for CMG and CEA. A solid and cystic tumor had strongly positive results for AAT and vimentin and focally positive results for CMG and pancreatic hormones. Microcystic adenomas had abundant glycogen and strong immunoreactivity for cytokeratin. Ductal cell carcinomas showed cells focally positive for AAT, lysozyme, CMG, CA 19-9, and CEA. CONCLUSIONS: The localization of reg protein was not consist with that of any other proteins examined in the current study. Thus, reg protein was considered a useful marker for acinar cell differentiation; however, ectopic expression of reg also was observed in ductal cell carcinomas. In ductal cell carcinomas, expression of reg immunoreactivity was considered as one of phenotypic heterogeneity, as seen in AAT, lysozyme, and CMG immunoreactivity.
The effects of E. coli L-asparaginase on cultured human pancreatic carcinoma (MIA PaCa-2) have been studied. The enzyme (1 U/ml) inhibited growth and protein synthesis in both MIA PaCa-2 and PANC-1, another pancreatic carcinoma cell line, but had little or no effect on human breast carcinoma or melanoma cells. The inhibition of protein synthesis by E. coli L-asparaginase was largely reversed by L-glutamine but not by L-asparagine. The growth of both MIA PaCa-2 and PANC-1 showed absolute dependence on L-glutamine. These results indicate that the effect of E. coli L-asparaginase on cultured pancreatic carcinoma cells is exerted at least in part through its L-glutaminase activity. Although the addition of L-glutamine to the culture appeared to prevent cell death caused by L-asparaginase, it did not restore the ability of the cells to proliferate. Asparaginase derived from vibrio succinogenes, which is virtually free of L-glutaminase activity, was equally inhibitory to MIA PaCa-2 cell growth but did not affect protein synthesis. It is concluded that the inhibition of growth of cultured pancreatic carcinoma cells by E. coli asparaginase is a combined function of both its L-asparaginase and L-glutaminase activity.
Intracellular thiols have been proposed as mediators of resistance to alkylating agents and cisplatin. As metallothionein is the predominant protein thiol, we examined its relationship to cisplatin resistance in human ovarian cancer cell lines. A human ovarian carcinoma cell line, A2780, derived from an untreated patient, was treated with cisplatin in several ways and the induced resistance to cisplatin ranged from 13- to 68-fold. The degree of resistance was dependent upon the method of selection. The drug-resistant cell lines also developed low levels of cross-resistance to cadmium. Additional cell lines established from untreated patients or ovarian cancer patients refractory to cisplatin- and/or carboplatin-containing combination chemotherapy were studied. The most cisplatin-resistant cell lines, OVCAR-8 and -10, were from patients previously treated with intensive chemotherapy. OVCAR-8 was relatively cross-resistant to cadmium while OVCAR-10 appeared relatively sensitive. Cell lines were examined for expression of metallothionein mRNA to evaluate the relationship between cisplatin resistance, cadmium cross-resistance and metallothionein expression. Only two of the cell lines with in vitro-induced resistance to cisplatin, 2780E80 and 2780CP70B3, had detectable metallothionein mRNA. The other cell lines selected in vitro for cisplatin resistance, as well as the parental A2780 ovarian cancer cell line, showed no expression at our level of detection. There was variable expression of metallothionein among the OVCAR cell lines. Cell lines from untreated patients, OVCAR-5 and -7, did express metallothionein, while the most cisplatin-resistant cell lines, OVCAR-8 and -10, did not. We also examined cisplatin induction of metallothionein mRNA in the cell lines. Only 2780CP70B3 among the cell lines with in vitro-induced cisplatin resistance showed increased expression after short-term exposure to cisplatin. OVCAR-4 also had a slight increase in expression after exposure to cisplatin. Mouse C127 cells transfected with a bovine papilloma virus-metallothionein gene construct were compared for cisplatin sensitivity to the same cell type transfected with bovine papilloma virus alone. In this model system, metallothionein expression did not influence cisplatin cytotoxicity. On the basis of these studies, we conclude that there is no causal relationship between metallothionein expression and cisplatin resistance.
Human glandular kallikrein-1 gene (hGK-1) is closely related to the gene of human prostate-specific antigen (PSA) and both genes are expressed in the human prostate. We have studied PSA and hGK-1 mRNAs in human prostatic tissue samples from patients with benign prostatic hyperplasia (BPH) or adenocarcinoma (CA), using Northern and slot-blot analysis in order to gain insight into the expression of these highly similar genes. Multiple mRNAs were found to originate from both genes. The major mRNA species of 1.6 kb accounted for 57% to 76% of the total coding capacity for PSA in different tissue specimens, but a variant mRNA species of 1.9 kb was also abundant. Most of the BPH samples contained marked amounts of an aberrant 0.9 kb mRNA, and long PSA mRNAs of 6.1 kb, 4.5 kb and 3.1 kb were found in elevated amounts in some of the CA samples. The amount of PSA mRNAs that would produce aberrant PSA proteins if translated into protein varied from 18% to 38% in these tissue samples. The major mRNA species originating from hGK-1 was of 1.6 kb, but other less abundant mRNA species could also be observed. The amount of PSA and hGK-1 mRNAs was determined from slot blots hybridized with specific oligonucleotide probes. No significant differences could be found in the PSA gene expression between BPH and CA samples. The total amount of the PSA mRNAs in all the different BPH specimens was fairly similar, but there was a 3-fold difference between the highest and lowest PSA mRNA levels in the CA samples. The hGK-1 mRNA levels in the BPH specimens studied demonstrated greater variance than the PSA mRNA levels in the same samples. The correlation between PSA gene and hGK-1 expression in the BPH samples was good, suggesting that there are similarities in the regulation of these genes. However, the lack of correlation between the amounts of PSA and hGK-1 mRNAs in the CA samples except in sample C1 indicates that there are also differences in the gene regulation. The observed 3.7- to 6.5-fold excess of PSA mRNAs as compared with the amount of hGK-1 mRNAs present in the same tissue specimen also indicated differences in the cis- or trans-acting regulatory elements of these genes.
Molecular characterization of HLA-class-I expression was investigated in human tumor cell lines at the protein and mRNA levels using locus-specific monoclonal antibodies (MAbs) and probes. Some cell lines exhibited a differential expression of HLA-A and HLA-B products and also showed differences in the inducibility of HLA-class-I genes by gamma-IFN. Thus, gamma-IFN stimulation induced predominantly HLA-B mRNA in the HeP-2 cell line, which showed imbalances in basal levels of HLA-A and HLA-B expression. This unequal inducibility of HLA genes may imply that locus-specific regulatory mechanisms are involved in the expression of individual HLA products. The specific mechanism controlling the differential expression of HLA subsets appears to be independent of c-myc activity. Northern blot analysis found no relationship between c-myc mRNA levels and specific mRNA for HLA-A and HLA-B antigens.
We have studied the effects of 8 cytokines and their combinations on the in vitro growth of 10 human small-cell cancer lines (SCLC). Interferon-alpha and gamma (IFN-alpha and gamma) caused significant but slight growth inhibition over a 7-day incubation period. However, none of the other 6 cytokines, tumor necrosis factor (TNF), lymphotoxin (LT), interleukin-1 beta (IL-1 beta) interleukin-2 (IL-2), transforming growth factor-beta 1 (TGF-beta 1), or granulocyte colony-stimulating factor (G-CSF), modified SCLC cell proliferation. In contrast, all 10 lines were sensitive to lysis by natural killer (NK) and lymphokine-activated killer (LAK) cells. Sensitivity to LAK cells could be increased by pretreatment of SCLC cells with IFN-gamma. As resistance to the cytostatic/cytotoxic activity of some cytokines has been associated with autocrine production of cytokines, we screened the SCLC lines for cytokine mRNAs. Within the limits of detection of the assay we found no expression of TNF, TGF-beta 1, IL-1 beta or IL-6 mRNA in the 10 SCLC lines.
Localization of nucleolar protein B23 in HL-60 cells under the treatment by iron chelator deferoxamine (DSF) was studied using indirect immunofluorescence. Bright nucleolar fluorescence was observed in exponentially growing control cells. The addition of DSF in the culture system resulted in time- and dose-dependent induction of protein B23 translocation from nucleoli to nucleoplasm, inhibition of cell growth, DNA and RNA synthesis. The addition of FeCl3 at culture initiation completely reversed the effects of DSF. Furthermore, significant numbers of HL-60 cells could be rescued from the effects of DSF when iron was added back as late as 24 hr after exposure to DSF. Cells resumed their abilities to grow and to synthesize DNA and RNA upon the iron rescue. Protein B23, accordingly, relocated from nucleoplasm to nucleoli. These results indicate the importance of iron for proliferation of leukemic cells and localization of protein B23 in nucleoli. Preribosomal ribonucleoprotein particles (pre-rRNPs) were extracted from isolated nucleoli of HL-60 cells and fractionated on sucrose density gradients. Protein B23 was found to be co-localized with the pre-rRNPs as determined by ELISA assays. No such B23-associated pre-rRNPs or other pre-rRNP fractions were obtained from nucleoli of DSF-treated cells. These results suggest that one of the effects of the anti-proliferative action of DSF is the inhibition of rRNA synthesis in nucleoli. Due to the lack of new synthesis of rRNA in nucleoli, protein B23 loses its binding target and translocates into the nucleoplasm. B23 translocation, as observed by immunofluorescence, may be a simple and rapid method for assessing inhibition of cell growth in response to anti-proliferative drugs such as deferoxamine in cancer chemotherapy.
The effects of EGF and TGF-beta 1 on the proliferation of 2 ovarian carcinoma cell lines (IGROV1 and OVCCR1) were evaluated. The cell lines were adapted to grow in a restricted serum (0.5%) medium. EGF was required for proliferation of both ovarian cell lines. Low doses of TGF-beta 1 inhibited clonogenic capacity and attenuated the EGF-mediated stimulation of DNA synthesis in OVCCR1 cells. TGF-beta 1 inhibited OVCCR1 cell proliferation by blocking the cell cycle at the G1/S transition. TGF-beta 1 did not affect either clonal or monolayer growth of IGROV1 cells. Both cell lines express type-I and type-III TGF-beta receptors, suggesting that the unresponsiveness of IGROV1 cells to TGF-beta 1 occurs at a post-receptor level. TGF-beta 1 mRNA was detected in OVCCR1 cells and in 8 out of 11 of the ovarian tumor specimens examined.