Risk factors for transmission of tuberculosis among United States-born African Americans and Whites.

SETTING: Tuberculosis (TB) patients and their contacts enrolled in nine states and the District of Columbia from 16 December 2009 to 31 March 2011. OBJECTIVE: To evaluate characteristics of TB patients that are predictive of tuberculous infection in their close contacts. DESIGN: The study population was enrolled from a list of eligible African-American and White TB patients from the TB registry at each site. Information about close contacts was abstracted from the standard reports of each site. RESULTS: Close contacts of African-American TB patients had twice the risk of infection of contacts of White patients (adjusted risk ratio [aRR] 2.1, 95%CI 1.3-3.4). Close contacts of patients whose sputum was positive for acid-fast bacilli on sputum smear microscopy had 1.6 times the risk of tuberculous infection compared to contacts of smear-negative patients (95%CI 1.1-2.3). TB patients with longer (>3 months) estimated times to diagnosis did not have higher proportions of infected contacts (aRR 1.2, 95%CI 0.9-1.6). CONCLUSION: African-American race and sputum smear positivity were predictive of tuberculous infection in close contacts. This study did not support previous findings that longer estimated time to diagnosis predicted tuberculous infection in contacts.

Mycobacterium tuberculosis bacteraemia: experience from a non-endemic urban centre.

The isolation of Mycobacterium tuberculosis from blood culture specimens has been associated with human immunodeficiency virus (HIV) co-infection with variable impact on tuberculosis (TB) mortality reported. The overwhelming majority of M. tuberculosis bacteraemia cases were described in developing countries. We present a nested case-control analysis of clinical, sociodemographic and behavioural risk factors in patients with positive M. tuberculosis blood cultures compared with patients with negative blood cultures from a 9-year population-based active TB surveillance study conducted in Houston, Texas. There were 42 patients with M. tuberculosis bacteraemia, 47 blood culture negative patients and 3573 patients for whom no mycobacterial blood culture was requested. HIV infection was more common in patients for whom a mycobacterial blood culture was requested (79.8% versus 15.1% p <0.001). Of the patients with M. tuberculosis bacteraemia, six were HIV negative or had no documentation of HIV status, including five with immunosuppressive conditions other than HIV. Patients with M. tuberculosis bacteraemia were more likely than patients with negative blood cultures to be deceased at diagnosis or to die while on TB therapy (50.0% versus 17.0%, p <0.01), to report men-who-have-sex-with-men behaviour (31.7% versus 13.0%, p 0.03), to have renal failure (28.6% versus 6.4%, p 0.01), and to have HIV RNA levels higher than 500 000 copies/mL (61.9% versus 17.2%, p ≤0.01). Requests for mycobacterial culture of blood specimens were more common in HIV-infected individuals, and the presence of M. tuberculosis bacteraemia was associated with a significant increase in mortality.

Can mycobacterial katG genetic changes in isoniazid-resistant tuberculosis influence human disease features?

BACKGROUND: Isoniazid-resistant (INHr) Mycobacterium tuberculosis isolates often have katG mutations, and katG is a virulence factor in animal models. It is unclear if katG mutations or other mutations influence the characteristics of human disease. OBJECTIVE: To determine if the presence of INHr-conferring mutations were associated with distinct clinical features of tuberculosis (TB). METHODS: In a retrospective case-control study, INHr-conferring mutations were determined by DNA sequencing. We examined associations between clinical characteristics in patients with INHr M. tuberculosis (stratified by groups of relevant INHr-conferring mutations, including katG-S315T and inhA-C(-)15T mutations) and pan-susceptible (PS) isolates. RESULTS: Twenty-nine INHr TB cases and 50 PS controls were evaluated. Disease characteristics were not statistically different between INHr and PS cases. However, patients infected with non-katG mutants were associated with a higher rate of sputum culture conversion at 1 month after adjustment for relevant covariates (adjusted OR [aOR] 4.4, 95%CI 1.1-23.6, P = 0.04). Patients infected with katG mutants were associated with a higher rate of unilateral disease (aOR 4.7, 95%CI 1.0-34.3, P = 0.05). CONCLUSIONS: Most INHr TB cases with non-katG mutations have disease associated with faster response to treatment, and most cases with katG mutants have localized lung involvement.

Extra-pulmonary manifestations in a large metropolitan area with a low incidence of tuberculosis.

BACKGROUND: The increases in extra-pulmonary tuberculosis (EPTB) have been largely due to human immunodeficiency virus co-infection. The rates of EPTB have remained constant despite the decline in pulmonary tuberculosis (PTB) cases. OBJECTIVE: To evaluate covariates associated with EPTB. METHODS: A 4-year cohort of EPTB patients was compared with PTB cases. Enrollees were assessed for TB risk, medical records were reviewed, and Mycobacterium tuberculosis isolates were fingerprinted. RESULTS: We identified 538 EPTB cases (28.6%) in a total of 1878 enrollees. The most common sites of infection were lymph nodes (43%) and pleura (23%). EPTB cases included 320 (59%) males, 382 (71%) patients were culture-positive, and 332 (86.9%) patient isolates were fingerprinted. Fewer EPTB than PTB patients belonged to clustered M. tuberculosis strains (58% vs. 65%; P = 0.02). A multivariate model identified an increased risk for EPTB among African Americans (OR = 1.9, P = 0.01), HIV-seropositive (OR = 3.1, P < 0.01), liver cirrhosis (OR = 2.3, P = 0.02), and age <18 years (OR = 2.0, P = 0.04). Patients with concomitant pulmonary and extra-pulmonary infections were more likely to die within 6 months of TB diagnosis (OR = 2.3, P < 0.01). CONCLUSIONS: African American ethnicity is an independent risk factor for EPTB. Mortality at 6 months is partly due to the dissemination of M. tuberculosis and the severity of the underlying co-morbidity.

Extrathoracic tuberculosis lymphadenitis in adult HIV seronegative patients: a population-based analysis in Houston, Texas, USA.

OBJECTIVE: To evaluate the covariates associated with extrathoracic tuberculosis lymphadenitis (ETBL) among adult HIV-seronegative patients. METHODS: Enrollees were interviewed for TB risk assessment, their medical records were reviewed, and their Mycobacterium tuberculosis isolates underwent molecular characterization. Between 1 October 1995 and 30 September 1999, HIV-negative patients with ETBL were compared with other HIV-negative TB patients. RESULTS: We identified 73 ETBL cases (5%) out of a total of 1371 adult HIV-negative enrollees. Significant variables predicting ETBL in the univariate analysis included age < 45 years, female sex, Asian ethnicity, foreign birth, BCG vaccination, and infection with a M. tuberculosis isolate identified in major genetic group 1. Further analysis by birth country revealed increased ETBL risk for persons from countries other than the Americas and with a TB incidence > 25 per 100 000 per year. The multivariate model demonstrated increased risk for ETBL for patients of female sex (OR = 2.6, P < 0.01) and birth in Africa or South-east Asia (OR = 4.8; P = 0.03 and OR = 33.6; P = 0.01, respectively). CONCLUSIONS: In adult HIV-negative patients, ETBL occurs more frequently in females and in immigrants from countries other than the Americas; persons from India, South-east Asia and the Eastern Mediterranean exhibited the highest risk among these regions.

5' dinucleotide repeat polymorphism of NRAMP1 and susceptibility to tuberculosis among Caucasian patients in Houston, Texas.

SETTING: Houston Tuberculosis Initiative (HTI) and Baylor College of Medicine, Houston, Texas. OBJECTIVE: To further explore the association between the polymorphisms of NRAMP1 and human susceptibility/resistance to tuberculosis (TB), specifically to determine whether the reported association shown for blacks and Asians holds true for Caucasian populations. DESIGN: In a case-control study, 135 adult Caucasian TB patients and 108 adult Caucasian HIV-seronegative non-TB controls were analyzed for the association between the polymorphisms in NRAMP1 gene and clinical TB. RESULTS: Heterozygote at 5'(GT)n, a dinucleotide repeat polymorphism in the promoter of NRAMP1, was observed at significantly higher frequencies among HIV-negative patients with pulmonary TB (41.6%; OR 2.02; 95%CI 1.11-3.64), extra-pulmonary TB (66.7%; OR 4.80; 95%CI 1.34-17.15), and HIV-seropositive TB patients (50%; OR 3.77; 95%CI 1.33-10.66) in comparison with the controls (27.8%). Homozygotes (GT)(10,10) were over-represented among HIV-positive TB patients (18.2%; OR 6.86; 95%CI 1.55-30.21) compared to the controls (5.5%). CONCLUSION: These findings suggest that the 5'(GT)n polymorphism of NRAMP1 modifies TB susceptibility in this Caucasian population, and could possibly be related to the site of infection among HIV-negative individuals and HIV-coinfected TB.

The contribution of ethnicity to Mycobacterium tuberculosis strain clustering.

OBJECTIVE: Descriptive study of molecular epidemiologic patterns of tuberculosis cases among ethnic minorities in Houston, Texas. DESIGN: Population-based, prospective, active surveillance, and molecular epidemiology study. PATIENTS: Tuberculosis cases reported to the City of Houston Tuberculosis Control Office between October 1995 and September 1998. RESULTS: During the study period, 1,139 culture-positive patients were enrolled for whom isolates of their culture specimen were available. Of these, 910 were part of an ethnic minority. Molecular characterization identified 689 of 1,139 isolates to be clonally related. Factors significantly associated with tuberculosis strain clustering in a multivariable logistic regression analysis were: birth in the United States, a history of homelessness, infection with the human immunodeficiency virus (HIV), pulmonary disease, infection with a tuberculosis strain from principal genetic group 1 or 3, living in a residence with five or more persons present, and use of public transportation more than once weekly. Asian ethnicity and increasing age were associated with decreased odds of clustering. CONCLUSIONS: Ethnicity was not a significant covariate for strain clustering after adjustments for factors related to socio-economic status.

Retroviral delivery of DNA into the livers of transgenic mice bearing premalignant and malignant hepatocellular carcinomas.

To develop gene therapy for hepatocellular carcinoma (HCC), we infused mice through the portal vein with retrovirus carrying the Escherichia coli beta-galactosidase reporter gene under the transcriptional control of the viral long terminal repeat (LTR) and the promoter from the mouse multidrug resistance gene mdr1b. Two transgenic mouse HCC models were used, one bearing the human hepatitis B viral envelope protein and the other SV40 T antigen. These animals develop HCC with predictable pathological manifestations. The viral transduction efficiency appeared to depend upon the stage of the disease in the animals. The most efficient transduction occurred when the livers had developed microscopic nodular hyperplasia; in some cases as many as 0.01-0.1 copies/cell were transduced. The transduction efficiency was lower in the late stage of the disease when livers had a heavy tumor burden and in the early stage when no lesion was evident. Low viral transduction efficacy was also seen in nontransgenic animals but was significantly increased by partial hepatectomy. The expression of the reporter gene in these animals was very low, as determined by histological staining. These results suggest that hepatocarcinogenesis can enhance retroviral delivery of foreign genes into the liver. Further development by increasing the viral transducing efficiency and the level of expression of transduced gene is required.

Two multidrug-resistant Friend leukemic cell lines selected with different drugs exhibit overproduction of different P-glycoproteins.

Two Friend leukemic multidrug-resistance (MDR) cell lines were established by exposure to stepwise increased concentrations of rhodamine-123 (RHO) (cell line RR-30) or Adriamycin (ADR) (cell line ARN-15). RR-30 displays preferential resistance to RHO, whereas ARN-15 is more resistant to ADR. The levels of resistance to other MDR drugs and reversibility by verapamil between these two MDR cell lines were somewhat different. Southern blot, RNase protection, and Western blot analysis using gene-specific probes demonstrated that RR-30 and ARN-15 cells preferentially amplified the mdr1 and mdr3 genes, respectively, leading to overexpression of the corresponding P-glycoproteins (p-gp). Our results suggest that members of the mdr gene family can be amplified independently by using different selecting agents, which could be responsible for the differences in the sensitivities to these selecting agents as well as to these MDR drugs.

Activation of distinct multidrug-resistance (P-glycoprotein) genes during rat liver regeneration and hepatocarcinogenesis.

The multidrug transporter P-glycoproteins are encoded by three multidrug-resistance (mdr) genes in rodents, designated mdr1a (mdr3), mdr1b (mdr1), and mdr2. Only the first two genes are functionally related to multidrug resistance. Activation of rodent mdr genes during liver regeneration and hepatocarcinogenesis has been reported. In mice, mdr1a is activated in hepatocellular carcinomas (HCCs) produced by various carcinogenic protocols, whereas both mdr1a and mdr2 are activated during liver regeneration. In this communication, we report isolating three gene-specific probes for the rat mdr homologues, which were used as probes in an RNase protection assay to demonstrate that mdr1b mRNA was expressed in HCCs induced by two different protocols. Furthermore, high levels of hepatic mdr1b mRNA but only moderate levels of mdr1a and mdr2 mRNA were seen in preneoplastic lesions in rats treated with 2-acetylaminofluorene. Likewise, highly elevated levels of hepatic mdr1b mRNA but only moderately increased levels of mdr1a and mdr2 mRNA were seen after partial hepatectomy. Nevertheless, the general patterns of tissue-specific expression of these three mdr genes were similar in rats and mice. These results reveal a complex hepatic gene expression pattern during hepatocarcinogenesis and hepatic proliferation for this conserved gene family in rodents.

Activation of multidrug resistance (P-glycoprotein) mdr3/mdr1a gene during the development of hepatocellular carcinoma in hepatitis B virus transgenic mice.

The expression of multidrug resistance (mdr) genes was investigated in the livers of transgenic mice that express the human hepatitis B virus large envelope polypeptide under the transcriptional control of a liver-specific promoter. These mice develop a storage disease due to the accumulation of a nonsecretable form of hepatitis B surface antigen in the hepatocyte. Liver cell injury is followed by a hepatocellular proliferative response, dysplasia, microscopic nodular hyperplasia, and finally hepatocellular carcinoma. The expression of mdr1, mdr2, and mdr3 genes was analyzed in livers at different stages of the disease by RNase protection assay, Western blot, and immunohistochemistry. RNase protection assay revealed that mdr3 mRNA expression was moderately increased in tissue with microscopic nodular hyperplasia and significantly overexpressed in hepatocellular carcinoma but undetectable in earlier stages of the disease. Western blot using isoform-specific anti-mdr3 antibody demonstrated that the expression of mdr3 protein reflected the steady-state level of mdr3 mRNA. Immunohistochemical analyses using anti-mdr3 isoform-specific antibody and monoclonal antibody C219, which recognizes all the three mdr isoforms, demonstrated selective overexpression in preneoplastic foci during the stage of microscopic nodular hyperplasia as well as in neoplastic hepatocytes in hepatocellular carcinoma. No consistent activation of mdr1 and mdr2 (but occasional coactivation with mdr1) genes during hepatocarcinogenesis was observed. Our results suggest that the hepatocellular mdr3-specific activation mechanism is associated with the late events of hepatocarcinogenesis in this model. The predictable kinetics of mdr gene expression in this transgenic tumor model suggest that it is suitable for future studies of the mechanism of mdr gene activation and the possible pharmacological consequences for mdr3 gene expression of hepatocellular carcinoma.

Postnatal development of organic cation transport and mdr gene expression in mouse kidney.

The apical surface of the proximal tubular epithelium is the site of both P-glycoprotein localization and postulated active secretion of organic cations in the mammalian kidney. P-glycoprotein has been shown to act as a pleiotropic drug efflux pump across the cell membrane of tumor cells expressing the multidrug resistance phenotype, whereas the renal organic anion and organic cation secretory systems serve the function of pleiotropic drug transport across the proximal tubule epithelium. Because most known substrates for P-glycoprotein are organic cations, we tested the hypothesis that the physiological function of this protein in the kidney is to mediate renal organic cation secretion. In one approach, we compared the postnatal development of organic cation transport with that of kidney mdr gene expression. Cimetidine-sensitive uptake of classical substrates for renal secretion (N-methyl nicotinamide and tetraethylammonium) into kidney slices developed gradually in neonate mice, reaching adult capacity in 4 to 6 weeks. P-glycoprotein and its mRNA, as estimated by immunohistochemical methods and RNAse protection analysis, were undetectable at birth and were expressed abruptly at the adult level between 2 and 3 weeks of age. In another approach, classical inhibitors of renal organic cation secretion (cimetidine and cyanine 863) failed to reverse resistance to adriamycin in Chinese hamster ovary and P388 cell lines, which possess the phenotypic traits of multidrug resistance. These results suggest that the cimetidine-sensitive component of organic cation secretion is mediated by a protein other than the P-glycoprotein in the mammalian kidney.

Structural and functional analyses of the promoter of the murine multidrug resistance gene mdr3/mdr1a reveal a negative element containing the AP-1 binding site.

We have previously shown that the multidrug-resistance/P-glycoprotein gene, mdr3/mdr1a, is activated in mouse hepatocellular carcinomas (HCC). In this study, we show that in a number of HCC-derived cell lines (Hepa1c1c, Hepa1c1c-BprC1, and Hepa1-6) mdr3 is expressed at high levels. To investigate transcriptional regulation of mdr3 in these cells, we have isolated a DNA fragment containing the 5' portion of the mouse mdr3 gene and performed a functional analysis of its promoter. Transient transfection assays using various lengths of the promoter sequence to direct expression of the chloramphenicol acetyltransferase (CAT) reporter gene revealed that the sequence located -94 nucleotides upstream from mouse mdr3 transcription start site functions as a negative element in mouse hepatoma cells. A canonical AP-1 binding sequence TGA-GTCA located at -117 is at least in part responsible for the negative effect from the following observations: (i) Alteration of this AP-1 sequence by site-directed mutagenesis enhanced CAT expression. (ii) Expression of CAT reporter gene was elevated when double-stranded DNA containing the AP-1 sequence, but not mutated sequences, was used as a competitor in cotransfection experiment. (iii) Enhancement of the CAT expression was also seen in cotransfection experiments using recombinant plasmid DNA expressing the c-jun/c-fos proteins, which interact with AP-1 sequences. Interestingly, the proximal region of the hamster pgp1 promoter shares striking sequence similarity with that of the mouse mdr3 gene, including the AP-1 site, but the AP-1 site in the hamster promoter serves as a positive regulator. Although previous studies have demonstrated that positive and negative transcription factors can modulate gene expression through interactions with c-jun/c-fos, this is the first study to show that an AP-1 site functions as a negative cis-element in the regulation of gene expression.

Analysis of the Chinese hamster P-glycoprotein/multidrug resistance gene pgp1 reveals that the AP-1 site is essential for full promoter activity.

Recent studies have revealed that the expression of P-glycoprotein/multidrug resistance genes is crucial for the development of resistance to a number of lipophilic cancer chemotherapeutic agents. To better understand the regulatory mechanisms of pgp gene expression, we isolated and characterized a DNA fragment containing the 5' portion of a Chinese hamster pgp gene. DNA sequence analysis revealed that this gene is pgp1, the hamster homologue of murine mdr3/mdr1a. This gene is expressed at a higher level in intestines than in kidney and liver, consistent with the expression pattern for the murine mdr3/mdr1a gene. The major transcription start site, determined by the S1 nuclease protection, RNase protection, and primer extension methods, lies 67 nucleotides upstream of the murine and human downstream transcription start site. A chimera containing 101 base pairs upstream from this start site and the chloramphenicol acetyltransferase (CAT) gene was able to direct CAT expression in transient transfection experiments. The AP-1 site, located at -48 base pairs, was crucial for the full pgp1 promoter activity, as demonstrated by site-directed mutagenesis of this site, enhancement of the CAT expression by cotransfection with the expression vectors encoding c-Jun/c-Fos genes, but sequestration with those containing retinoic acid receptor genes. The sequestration effect could be partially abolished when c-Jun/c-Fos genes were also included in cotransfection. An AP-1 or AP-1-like site is also present at the same location in both human and mouse mdr homologues. The involvement of AP-1 in the expression of mammalian pgp1-class genes is discussed.

Murine mdr-1, mdr-2, and mdr-3 gene expression: no coinduction with the Cyp1a-1 and Nmo-1 genes in liver by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Multidrug-resistance (MDR) genes are induced in the liver of rodents treated with a variety of foreign chemicals and hepatocarcinogens. It has been reported that 2,3,6,7-tetrachlorodibenzo-p-dioxin (TCDD) might increase hepatic MDR transcripts in the Fischer rat and the C57BL/6 (B6) inbred mouse strain having the high-affinity aromatic hydrocarbon (Ah) receptor, but not in the DBA/2 (D2) strain having the low-affinity Ah receptor. These intriguing results suggest that TCDD might activate MDR gene expression by way of an Ah receptor-mediated signal transduction pathway. We have attempted to confirm these data in four inbred mouse strains: two (B6 and BALB/c) having the high-affinity Ah receptor, and two (D2 and AKR) having the low-affinity Ah receptor. The RNase protection assay was used to distinguish between the MDR1, MDR2, and MDR3 mRNAs. TCDD treatment at high (100 micrograms/kg) and low (1 mu/kg) doses, a time course from 6 to 96 hr of TCDD treatment, progeny from the B6D2F1 x D2 backcross, and transcriptional run-on experiments were performed. The Cyp1a-1 (cytochrome P1450) and Nmo-1 [NAD(P)H:menadione oxidoreductase] genes, two members of the TCDD-inducible [Ah] battery, were used as positive controls. We were unable to detect significant coinduction of MDR1, MDR2, or MDR3 mRNA with CYP1A1 mRNA or with Cyp1a-1 or Nmo-1 transcription under any conditions. Therefore, we conclude that any effects that TCDD might have on MDR expression must be substantially different from TCDD effects on genes known to be induced via the Ah receptor.

Coordinate activation of multidrug-resistance (P-glycoprotein) genes mdr2 and mdr3 during mouse liver regeneration.

We previously reported that only one of the three mouse multidrug-resistance (mdr) genes, mdr3, is activated in hepatocellular carcinomas (HCCs). The present study examined the expression of mdr family members during mouse liver regeneration after partial hepatectomy to determine whether the regeneration that occurs during hepatic tumorigenesis is responsible for mdr3 elevation in HCC. We demonstrated that in both C3H/HeN and B6C3/F1 mice strains, the levels of both mdr2 and mdr3 mRNAs coordinately increased five- to sevenfold 24 h after partial hepatectomy, whereas the levels of mdr1 mRNA were not statistically different from those in the controls. Forty-eight hours after partial hepatectomy, mdr mRNA levels decreased and in most cases returned to normal levels after 72 h. These results indicate that mdr3 induction during hepatocarcinogenesis is not due to liver regeneration alone.

Overexpression of the multidrug resistance gene mdr3 in spontaneous and chemically induced mouse hepatocellular carcinomas.

Overexpression of a family of plasma membrane glycoproteins, known as P-glycoproteins, is commonly associated with multidrug resistance in animal cells. In rodents, three multidrug resistance (mdr or pgp) genes have been identified, but only two can confer the multidrug resistance phenotype upon transfection into animal cells. Using the RNase protection method, we demonstrated that the levels of three mdr gene transcripts differ among mouse tissues, confirming a previous report that the expression of these genes is tissue specific (J.M. Croop, M. Raymond, D. Huber, A. DeVault, R. J. Arceci, P. Gros, and D. E. Housman, Mol. Cell. Biol. 9:1346-1350, 1989). The levels of mdr transcripts were determined for mouse liver tumors spontaneously arising in both C3H/HeN and transgenic animals containing the hepatitis B virus envelope gene and for tumors induced by two different carcinogenic regimens in C57BL/6N and B6C3-F1 mice. The mdr3 gene was overexpressed in all 22 tumors tested. Our results demonstrate that overexpression of the mdr3 gene in mouse liver tumors does not require exposure of the animals to carcinogenic agents and suggest that its overexpression is associated with a general pathway of hepatic tumor development. The overexpression of the mdr3 gene, which is the homolog of human mdr1 gene, in hepatocellular carcinomas may be responsible for the poor response of these tumors to cancer chemotherapeutic agents.

Model for the formation of double minutes from prematurely condensed chromosomes of replicating micronuclei in drug-treated Chinese hamster ovary cells undergoing DNA amplification.

Double minutes (DM) have been associated with gene amplification in drug-resistant cells and tumor cells. However, the mechanisms by which DM are formed have not been elucidated. We present here a model to describe a possible mechanism of DM formation based on the observations made in two independent early drug-selected multidrug-resistant cell lines and from in vitro somatic cell fusion experiments between synchronized S- and M-phase cells. The multidrug-resistant cell lines contain both DM and amplified mdr (P-glycoprotein) gene. Cytogenetic analyses of cells at early stages of selection revealed the presence of a number of micronuclei in a subpopulation of these cells. These micronuclei were often asynchronous in their progression through the cell cycle. As a result, premature condensation of micronuclear chromatin was often observed in metaphase plates. The pulverized chromatin pattern seen in certain instances of S-phase prematurely condensed chromosomes displays a striking resemblance to DM structures. These DM-like structures are linked by replicating DNA as revealed by DNA labeling experiments. Somatic cell hybrids between S- and M-phase cells when grown in vitro demonstrated that S-phase prematurely condensed chromatin indeed gives rise to extra chromosomal structures in the successive cell generations. It is hypothesized that distinct DM-like structures may arise from the partially replicated and prematurely condensed S-phase chromosomes following their liberation as extra chromosomal entities after replication and/or recombination in the succeeding division cycle(s). The enrichment for DM containing specific genes in drug-resistant cells may result from the subsequent drug selections.

Specific gene amplification associated with consistent chromosomal abnormality in independently established multidrug-resistant Chinese hamster ovary cells.

Multidrug-resistant (MDR) Chinese hamster ovary (CHO) cell lines were established by selection for resistance to the toxicities of vinblastine (VB) and Adriamycin (AD) in progressively increasing drug concentrations. These cell lines have amplified the DNA sequence that has previously been shown to be amplified in another MDR CHO cell line which was selected with vincristine (VC). An overproduced 4.5 kb mRNA was detected in these MDR cell lines. We report here that the levels of DNA amplification and the 4.5 kb transcript do not correlate with the levels of drug resistance, suggesting that either translational control for the expression of the amplified gene is involved or multiple genes are participating in conferring drug resistance in these cell lines. The amplified DNA sequence was used as a probe and localized by in situ hybridization to chromosome 1q 26-28 (middle portion of the long arm) in the drug-sensitive CHO line, but proximal to the telomere of chromosome 1q in both VB- and AD-selected MDR cell lines. This is consistent with results that have been previously reported for the VC-selected MDR cell lines. Cytogenetic analyses revealed abnormal chromosomal banding patterns or homogeneously staining regions (HSR) between 1q 26-28 and the 1q ter in these independently established MDR lines. These results, taken together, suggest that chromosomal rearrangements leading to gene translocation have consistently accompanied gene amplification in these MDR cell lines. The mechanisms of translocation and its implication in multidrug resistance in these cell lines are discussed.

Multidrug-resistant phenotype cosegregates with an amplified gene in somatic cell hybrids of drug-resistant Chinese hamster ovary cells and drug-sensitive murine cells.

Gene amplification has been associated with multidrug resistance (MDR) in several drug-resistant Chinese hamster ovary (CHO) cell lines which exhibit cross-resistance to other unrelated, cytotoxic drugs. In situ hybridization studies (Teeter et al., J. Cell Biol., in press) suggested the presence of an amplified gene associated with the MDR phenotype on the long arm of either of the largest CHO chromosomes (1 or Z1) in vincristine-resistant cells. In this study, somatic cell hybrids were constructed between these vincristine-resistant CHO cells and drug-sensitive murine cells to determine the functional relationship between the chromosome bearing the amplified sequences and the MDR phenotype. Hybrids exhibited primary drug resistance and MDR in an incomplete dominant fashion. Hybrid clones and subclones segregated CHO chromosomes. Concordant segregation between vincristine resistance, the MDR phenotype, the presence of the MDR-associated amplified sequences, overexpression of the gene located in those sequences, and CHO chromosome Z1 was consistent with the hypothesis that there is an amplified gene on chromosome Z1 of the vincristine-resistant CHO cells which is responsible for the MDR in these cells. A low level of discordance between CHO chromosomes Z8 and 2 and the drug resistance phenotype suggests that these chromosomes may contain genes involved with the MDR phenotype.